Beclin-1

Macroautophagy is involved in the bulk degradation of long-lived cytosolic proteins and subcellular organelles, which is important for the survival of cells during starvation. To identify potential players of the autophagy process, we subjected HCT116 cells cultured in complete medium and in Earle's balanced salt solution to proteomics analysis. In approximately 1,500 protein spots detected, we characterized 52 unique proteins, whose expression levels were significantly changed following starvation. Notably, we found that Annexin A1 was significantly upregulated following starvation at both mRNA and protein levels. Inhibition of Annexin A1 expression with specific siRNA did not alter starvation-induced autophagy as measured by the level of lipidated LC3, but significantly reversed autophagy degradation as measured by the level of p62/SQSTM 1. Thus Annexin A1 seemed to be positively upregulated during starvation to promote autophagic degradation. Overall, the data presented in this study established a expression profile of the proteome in starved cells, which allowed the identification of proteins with potential signficance in starvation-induced autophagy.

Ovarian cancer survival and treatment have improved minimally in the past 20years. Novel treatment strategies are needed to combat this disease. This study investigates the effects of chemical inhibition of the CBFβ/RUNX protein-protein interaction on ovarian cancer cell lines. Ovarian cancer cell lines were treated with CBFβ/RUNX inhibitors, and the effects on proliferation, DNA replication, wound healing, and anchorage-independent growth were measured. RNA-Seq was performed on compound-treated cells to identify differentially expressed genes. Genes altered by compound treatment were targeted with siRNAs, and effects on DNA replication and wound healing were measured. Chemical inhibition of the CBFβ/RUNX interaction decreases ovarian cancer cell proliferation. Inhibitor treatment leads to an S-phase cell cycle delay, as indicated by an increased percentage of cells in S-phase, and a decreased DNA replication rate. Inhibitor treatment also reduces wound healing and anchorage-indepen...

Aims: Autophagy, the process by which cells break down spent biochemical and damaged components, plays an important role in cell survival following stress. High mobility group box 1 (HMGB1) regulates autophagy in response to oxidative stress. Results: Exogenous hydrogen peroxide (H 2 O 2) treatment or knockdown of the major superoxide scavenger enzyme, superoxide dismutase 1 (SOD1), by small interfering RNA (siRNA) increases autophagy in mouse and human cell lines. Addition of either SOD1 siRNA or H 2 O 2 promotes cytosolic HMGB1 expression and extracellular release. Importantly, inhibition of HMGB1 release or loss of HMGB1 decreases the number of autophagolysosomes and autophagic flux under oxidative stress in vivo and in vitro. Innovation: HMGB1 release may be a common mediator of response to oxidative stress. Conclusion: HMGB1 is important for oxidative stress-mediated autophagy and serves as a new target for the treatment of stressassociated disorders. Antioxid. Redox Signal. 15, 2185-2195.

Aims: Autophagy, the process by which cells break down spent biochemical and damaged components, plays an important role in cell survival following stress. High mobility group box 1 (HMGB1) regulates autophagy in response to oxidative stress. Results: Exogenous hydrogen peroxide (H 2 O 2) treatment or knockdown of the major superoxide scavenger enzyme, superoxide dismutase 1 (SOD1), by small interfering RNA (siRNA) increases autophagy in mouse and human cell lines. Addition of either SOD1 siRNA or H 2 O 2 promotes cytosolic HMGB1 expression and extracellular release. Importantly, inhibition of HMGB1 release or loss of HMGB1 decreases the number of autophagolysosomes and autophagic flux under oxidative stress in vivo and in vitro. Innovation: HMGB1 release may be a common mediator of response to oxidative stress. Conclusion: HMGB1 is important for oxidative stress-mediated autophagy and serves as a new target for the treatment of stressassociated disorders. Antioxid. Redox Signal. 15, 2185-2195.

The main energy source of cancer cells is known as glucose. However, they use glutamine as a second energy source besides glucose. In this study we investigated effect of siRNA mediated inhibition of glutaminase (GLS1) enzyme in the first step of glutamine metabolism on proliferation and apoptosis which are the main features of cancer. In our study we determined the cell viability by MTS analysis and the apoptosis rate by Annexin V using triple negative MDA-MB 231 cell line belonging to aggressive subtype of breast cancer. Our study demonstrated that siRNA-mediated silencing GLS1 reduced proliferation in this cancer cell line. It has been shown that inhibition of GLS1 significantly decreased cells, but there was no change in the rate of apoptosis. The cause of this decrease may be through a different pathway other than apoptosis. In this study we have shown that GLS1 inhibition does not induce apoptosis in this cell line, contrary to the literature, and activates the death pathway t...

Loss of growth inhibitory response to transforming growth factor-beta (TGF-beta) is a common feature of epithelial cancers. Recent studies have reported that genetic lesions and overexpression of oncoproteins in TGF-beta/Smads signalling cascade contribute to the TGF-beta resistance. Here, we showed that the overexpressed FOXG1 was involved in attenuating the anti-proliferative control of TGF-beta/Smads signalling in ovarian cancer. FOXG1 and p21(WAF1/CIP1) expressions were evaluated by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), western blot and immunohistochemical analyses. The effect of FOXG1 on p21(WAF1/CIP1) transcriptional activity was examined by luciferase reporter assays. Cell lines stably expressing or short hairpin RNA interference-mediated knockdown FOXG1 were established for studying the gain-or-loss functional effects of FOXG1. XTT cell proliferation assay was used to measure cell growth of ovarian cancer cells. Quantitative RT-PCR ...

Background: Autophagy is a process that cells use to degrade and recycle cellular proteins, however, the role of autophagy in kidney fibrosis remains largely unknown. Results: Autophagy is responsible for the intracellular degradation of type I collagen. Conclusion: Autophagy negatively regulates and prevents excess collagen accumulation in the kidney. Significance: Our findings implicate a novel role of autophagy as a cytoprotective mechanism against renal fibrosis. Autophagy is a highly conserved cellular process regulating turnover of cytoplasmic proteins via a lysosome-dependent pathway. Here we show that kidneys from mice deficient in autophagic protein Beclin 1 exhibited profibrotic phenotype, with increased collagen deposition. Reduced Beclin 1 expression, through genetic disruption of beclin 1 or knockdown by specific siRNA in primary mouse mesangial cells (MMC), resulted in increased protein levels of type I collagen (Col-I). Inhibition of autolysosomal protein degradation by bafilomycin A 1 also increased Col-I protein levels and colocalization of Col-I with LC3, an autophagy marker, or LAMP-1, a lysosome marker, whereas treatment with TFP, an inducer of autophagy, resulted in decreased Col-I protein levels induced by TGF-␤1, without alterations in Col-I ␣1 mRNA. Heterozygous deletion of beclin 1 increased accumulation of aggregated Col-I under nonstimulated conditions, and stimulation with TGF-␤1 further increased aggregated Col-I. These data indicate that Col-I and aggregated, insoluble procollagen I undergo intracellular degradation via autophagy. A cytoprotective role of autophagy is implicated in kidney injury, and we demonstrate that low-dose carbon monoxide, shown to exert cytoprotection against renal fibrosis, induces autophagy to suppress accumulation of Col-I induced by TGF-␤1. We also show that TGF-␤1 induces autophagy in MMC via TAK1-MKK3-p38 signaling pathway. The dual functions of TGF-␤1, as both an inducer of Col-I synthesis and an inducer of autophagy and Col-I degradation, underscore the multifunctional nature of TGF-␤1. Our findings suggest a novel role of autophagy as a cytoprotective mechanism to negatively regulate and prevent excess collagen accumulation in the kidney.

Aim: Acquired docetaxel (DOC) resistance of prostate cancer (PCa) is still a clinical problem. In addition to failure in chemotherapy treatment, it causes tumor recurrence. Therefore, novel and more effective compounds are needed in DOC-resistant PCa treatment. This study aimed to investigate the possible cytotoxic and cell death-inducing activities of thymoquinone (TQ), one of the main active components of Nigella sativa L., on DOC-resistant prostate cancer cells. Material and Methods: DOC-resistant PC3 cells (DOC-R/PC3) were developed by the continuous culture with increment concentrations of DOC (1-10 nM) until they improved their growth and division abilities. The cell viability was determined by MTT assay. The MuseTM Annexin V & Dead Cell kit was performed to detect apoptotic cell death. Autophagic vacuoles were observed by staining autophagic vacuoles. The levels of LC3I, LC3II and Beclin-1 proteins were investigated via western blot analysis. Results: TQ inhibited the viabili...

Mechanisms underlying the pathogenesis of high-grade serous epithelial ovarian cancers (HGSOC) are not yet well defined although key precursor cells have been identified (including fimbriated fallopian tube epithelium, FTSECs). Since iron is elevated in endometriotic cysts and the pelvic cavity, it is suggested that this source of redox-active iron may contribute to ovarian cancer pathogenesis. Specifically, sources of nontransferrin-bound iron (NTBI) within the pelvic cavity could arise from ovulation, retrograde menstruation, follicular fluid, or iron overload conditions (i.e., hemochromatosis). Herein, we investigated the cellular response of p53-inactivated and telomerase-expressing (immortalized) FTSECs (Pax8+/FoxJ1−) to NTBI (presented as ferric ammonium citrate (FAC), supplemented in media for >2 months) in order to assess its ability to promote the transition to a tumor-like phenotype; this cellular response was compared with immortalized FTSECs transformed with H-RasV12A...

Although iron is essential for cell survival, dysregulated levels can contribute to cancer development or even cell death. The underlying mechanisms mediating these events remain unclear. Herein, proteomic alterations are assessed in iron-treated ovarian cell lines using reverse phase protein array (RPPA) technology and potential functional responses via ingenuity pathway analysis (IPA). Using these approaches, upregulation of pathways modulating organismal death with alterations in mTOR, MAPK, and AKT signaling in HEY ovarian cancer cells in contrast to T80 non-malignant ovarian cells is noted. Since modulation of cell death is mediated in part via microphthalmia-associated transcription factor (MiTF) family, which regulates lysosomal biogenesis and autophagosome formation by upregulating expression of coordinated lysosomal expression and regulation (CLEAR) network, expression changes in these factors in response to iron are investigated. Increased transcription factor EB (TFEB) in...

Iron is an essential element required for many processes within the cell. Dysregulation in iron homeostasis due to iron overload is detrimental. This nutrient is postulated to contribute to the initiation of cancer; however, the mechanisms by which this occurs remain unclear. Defining how iron promotes the development of ovarian cancers from precursor lesions is essential for developing novel therapeutic strategies. In this review, we discuss (1) how iron overload conditions may initiate ovarian cancer development, (2) dysregulated iron metabolism in cancers, (3) the interplay between bacteria, iron, and cancer, and (4) chemotherapeutic strategies targeting iron metabolism in cancer patients.

Crocin, a bioactive molecule of saffron, inhibited proliferation of both HCT116 wild-type and HCT116 p53(-/-) cell lines at a concentration of 10 mM. Flow cytometric analysis of cell cycle distribution revealed that there was an accumulation of HCT116 wild-type cells in G1 (55.9%, 56.1%) compared to the control (30.4%) after 24 and 48 h of crocin treatment, respectively. However, crocin induced only mild G2 arrest in HCT116 p53(-/-) after 24 h. Crocin induced inefficient autophagy in HCT116 p53(-/-) cells, where crocin induced the formation of LC3-II, which was combined with a decrease in the protein levels of Beclin 1 and Atg7 and no clear p62 degradation. Autophagosome formation was not detected in HCT116 p53(-/-) after crocin treatment predicting a nonfunctional autophagosome formation. There was a significant increase of p62 after treating the cells with Bafilomycin A1 (Baf) and crocin compared to crocin exposure alone. Annexin V staining showed that Baf-pretreatment enhanced th...

Autophagy can lead to cell death in response to stress, but it can also act as a protective mechanism for cell survival. We show that TGF-␤1 induces autophagy and protects glomerular mesangial cells from undergoing apoptosis during serum deprivation. Serum withdrawal rapidly induced autophagy within 1 h in mouse mesangial cells (MMC) as determined by increased microtubule-associated protein 1 light chain 3 (LC3) levels and punctate distribution of the autophagic vesicle-associated-form LC3-II. We demonstrate that after 1 h there was a time-dependent decrease in LC3 levels that was accompanied by induction of apoptosis, evidenced by increases in cleaved caspase 3. However, treatment with TGF-␤1 resulted in induction of the autophagy protein LC3 while suppressing caspase 3 activation. TGF-␤1 failed to rescue MMC from serum deprivation-induced apoptosis upon knockdown of LC3 by siRNA and in MMC from LC3 null (LC3 ؊/؊) mice. We show that TGF-␤1 induced autophagy through TAK1 and Akt activation, and inhibition of PI3K-Akt pathway by LY294002 or dominant-negative Akt suppressed LC3 levels and enhanced caspase 3 activation. TGF-␤1 also up-regulated cyclin D1 and E protein levels while down-regulating p27, thus stimulating cell cycle progression. Bafilomycin A1, but not MG132, blocked TGF-␤1 down-regulation of p27, suggesting that p27 levels were regulated through autophagy. Taken together, our data indicate that TGF-␤1 rescues MMC from serum deprivationinduced apoptosis via induction of autophagy through activation of the Akt pathway. The autophagic process may constitute an adaptive mechanism to glomerular injury by inhibiting apoptosis and promoting mesangial cell survival.

Autophagy is a highly conserved cellular process regulating turnover of cytoplasmic proteins via a lysosome-dependent pathway. Here we show that kidneys from mice deficient in autophagic protein Beclin 1 exhibited profibrotic phenotype, with increased collagen deposition. Reduced Beclin 1 expression, through genetic disruption of beclin 1 or knockdown by specific siRNA in primary mouse mesangial cells (MMC), resulted in increased protein levels of type I collagen (Col-I). Inhibition of autolysosomal protein degradation by bafilomycin A1 also increased Col-I protein levels and colocalization of Col-I with LC3, an autophagy marker, or LAMP-1, a lysosome marker, whereas treatment with TFP, an inducer of autophagy, resulted in decreased Col-I protein levels induced by TGF-β1, without alterations in Col-I α1 mRNA. Heterozygous deletion of beclin 1 increased accumulation of aggregated Col-I under nonstimulated conditions, and stimulation with TGF-β1 further increased aggregated Col-I. The...

T315, an integrin-linked kinase (ILK) inhibitor, has been shown to suppress the proliferation of breast cancer, stomach cancer and chronic lymphocytic leukemia cells. Here we demonstrate that T315 decreases cell viability of acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells are less sensitive than leukemia cells to T315. T315 down regulates protein kinase B (Akt) and p-Akt and induces caspase activation, poly-ADP-ribose polymerase (PARP) cleavage, apoptosis and autophagy through an ILK-independent manner. Interestingly, pretreatment with autophagy inhibitors rescues cells from apoptosis and concomitant PARP cleavage, which implicates a key role of autophagic cell death in T315-mediated cytotoxicity. T315 also demonstrates efficacy in vivo, suppressing the growth of THP-1 xenograft tumors in athymic nude mice when administered intraperitoneally. This study shows that autophagic cell death and apoptosis cooperatively contribute to the anticancer activity of T315 in AML cells. In conclusion, the complementary roles of apoptotic and autophagic cell death should be considered in the future assessment of the translational value of T315 in AML therapy.

Sanguinarine (SNG), a natural compound with an array of pharmacological activities, has promising therapeutic potential against a number of pathological conditions, including malignancies. This research aimed to investigate the antiproliferative and anti-cancer potential of SNG against two well characterized papillary thyroid cancer (PTC) cell lines, BCPAP and TPC-1 .In both cell lines , SNG was able to inhibit cell proliferation in time and dose dependent manner. Western blot analysis revealed increased expression of apoptosis and autophagy markers , caspase-3,cleaved caspase-3 , P62, and LC3. SNG modulate its anticancer effect through ROS production, because NAC was able to reverse SNG effect. Interestingly, co-treatment of PTC with SNG and cisplatin amplified anticancer activity. Finally, SNG treatment of PTC spheroid suppressed its growth with downregulation of stemness markers including ALDH2 and SOX2 markers. In conclusion, SNG enhanced the anti cancer activity against PTC cel...

High-resolution array comparative genomic hybridization of 235 serous epithelial ovarian cancers demonstrated a regional increase at 3q26.2 encompassing SnoN/SkiL, a coregulator of SMAD/TGFβ signaling. SnoN RNA transcripts were elevated in ~80% of advanced stage serous epithelial ovarian cancers. In both immortalized normal (TIOSE) and ovarian carcinoma cell lines (OVCA), SnoN RNA levels were increased by TGFβ stimulation and altered by LY294002 and JNK II inhibitor treatment suggesting that the PI3K and JNK signaling pathways may regulate TGFβ-induced increases in SnoN RNA. In TIOSE, SnoN protein levels were reduced 15 minutes post TGFβ-stimulation, likely by proteosome-mediated degradation. In contrast, in OVCA, SnoN levels were elevated 3 hours post-stimulation potentially as a result of inhibition of the proteosome. To elucidate the role of SnoN in ovarian tumorigenesis, we explored the effects of both increasing and decreasing SnoN levels. In both TIOSE and OVCA, SnoN siRNA decreased cell growth between 20-50% concurrent with increased p21 levels. In TIOSE, transient expression of SnoN repressed TGFβ induction of PAI-1 promoters with little effect on the p21 promoter or resultant cell growth. In contrast to the effects of transient expression, stable expression of SnoN in TIOSE led to growth arrest through induction of senescence. Collectively, these results implicate SnoN levels in multiple roles during ovarian carcinogenesis: promoting cellular proliferation in ovarian cancer cells and as a positive mediator of cell cycle arrest and senescence in non-transformed ovarian epithelial cells.

Objective: Glutamine metabolism is an important pathway in cell proliferation and tumor progression. The first enzyme to be converted in the process of glutamine metabolism, glutaminase 1 (GLS1), exhibits increased expression in many types of cancer, including breast cancer. Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with high glutamine metabolic activity. The aim of this research was to examine the effects on glutamine metabolism and carcinogenic properties following small interfering RNA (siRNA)-mediated inhibition of GLS1 in glutamine-dependent TNBC. Materials and Methods: The effects on cell proliferation, migration, apoptosis, colony formation, and the cell cycle of MDA-MB-231 cells using different siRNAs targeting GLS1 were analyzed using an MTS assay, a wound-healing assay, clonogenic analysis, and annexin V and propidium iodide staining methods. The protein expression of GLS1, integrin beta 1 (β1), caspase-3, and p21 were examined using western blot analysis and flow cytometry. Results: The findings revealed that cell viability, migration, and colony formation were significantly suppressed in MDA-MB-231 cells transfected with 2 different GLS1 siRNAs. Furthermore, the results of flow cytometry and western blot analysis demonstrated that knockdown of GLS1 induced arrest in the G0/G1 phase of the cell cycle through the p21 signaling pathway, but did not induce apoptosis. Conclusion: GLS1 is needed for cell proliferation and promotes tumor progression and growth of MDA-MB 231 cells. siRNAs may provide a means to downregulate GLS1 and offer a promising target for breast cancer therapy.

Autophagy is a self-digestion process that degrades intracellular structures in response to stresses leading to cell survival. When autophagy is prolonged, this could lead to cell death. Generation of reactive oxygen species (ROS) through oxidative stress causes cell death. The role of autophagy in oxidative stress-induced cell death is unknown. In this study, we report that two ROS-generating agents, hydrogen peroxide (H 2 O 2) and 2-methoxyestradiol (2-ME), induced autophagy in the transformed cell line HEK293 and the cancer cell lines U87 and HeLa. Blocking this autophagy response using inhibitor 3-methyladenine or small interfering RNAs against autophagy genes, beclin-1, atg-5 and atg-7 inhibited H 2 O 2 or 2-ME-induced cell death. H 2 O 2 and 2-ME also induced apoptosis but blocking apoptosis using the caspase inhibitor zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) failed to inhibit autophagy and cell death suggesting that autophagy-induced cell death occurred independent of apoptosis. Blocking ROS production induced by H 2 O 2 or 2-ME through overexpression of manganesesuperoxide dismutase or using ROS scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt decreased autophagy and cell death. Blocking autophagy did not affect H 2 O 2-or 2-ME-induced ROS generation, suggesting that ROS generation occurs upstream of autophagy. In contrast, H 2 O 2 or 2-ME failed to significantly increase autophagy in mouse astrocytes. Taken together, ROS induced autophagic cell death in transformed and cancer cells but failed to induce autophagic cell death in nontransformed cells.

Loss of growth inhibitory response to transforming growth factor-beta (TGF-beta) is a common feature of epithelial cancers. Recent studies have reported that genetic lesions and overexpression of oncoproteins in TGF-beta/Smads signalling cascade contribute to the TGF-beta resistance. Here, we showed that the overexpressed FOXG1 was involved in attenuating the anti-proliferative control of TGF-beta/Smads signalling in ovarian cancer. FOXG1 and p21(WAF1/CIP1) expressions were evaluated by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), western blot and immunohistochemical analyses. The effect of FOXG1 on p21(WAF1/CIP1) transcriptional activity was examined by luciferase reporter assays. Cell lines stably expressing or short hairpin RNA interference-mediated knockdown FOXG1 were established for studying the gain-or-loss functional effects of FOXG1. XTT cell proliferation assay was used to measure cell growth of ovarian cancer cells. Quantitative RT-PCR ...

Loss of growth inhibitory response to transforming growth factor-beta (TGF-beta) is a common feature of epithelial cancers. Recent studies have reported that genetic lesions and overexpression of oncoproteins in TGF-beta/Smads signalling cascade contribute to the TGF-beta resistance. Here, we showed that the overexpressed FOXG1 was involved in attenuating the anti-proliferative control of TGF-beta/Smads signalling in ovarian cancer. FOXG1 and p21(WAF1/CIP1) expressions were evaluated by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), western blot and immunohistochemical analyses. The effect of FOXG1 on p21(WAF1/CIP1) transcriptional activity was examined by luciferase reporter assays. Cell lines stably expressing or short hairpin RNA interference-mediated knockdown FOXG1 were established for studying the gain-or-loss functional effects of FOXG1. XTT cell proliferation assay was used to measure cell growth of ovarian cancer cells. Quantitative RT-PCR ...

Background Stanniocalcin 1 (STC1) is a secreted glycoprotein hormone. High expression of STC1 has been associated with several cancers including ovarian cancer, but its role in the development of ovarian cancer is not clear. Methods We used five human ovarian epithelial cancer cell lines (OVCA420, OVCA432, OVCA433, SKOV3, and HEY), immortalized human ovarian surface epithelial cells (T29 and T80), ovarian cancer tissues from 342 patients, serum from 73 ovarian cancer patients and from58 control subjects, and 116 mice, with six or eight per group. Protein expression was assessed. Cells overexpressing STC1 protein were generated by ectopic expression of human STC1 cDNA. STC1 expression was silenced by using small interfering RNA against STC1. Cell proliferation, migration, colony formation, and apoptosis were assessed. Xenograft tumor growth in mice was studied. Neutralizing anti-STC1 antibody was used to inhibit STC1 function. All statistical tests were two-sided. Results STC1 protein expression was higher in all human ovarian cancer cell lines examined than in immortalized human ovarian epithelial cell lines, higher in ovarian cancer tissue than in normal ovarian tissue (P < .001), and higher in serum from ovarian cancer patients than from control subjects (P = .021). Ovarian cancer cells with STC1 overexpression, compared with corresponding control cells, had increased cell proliferation, migration, and colony formation in cell culture and increased growth of xenograft tumors in mice. These activities in normal or malignant ovarian cells with STC1 overexpression, compared with control cells, were also accompanied by increased expression of cell cycle regulatory proteins and antiapoptotic proteins but decreased cleavage of several caspases. Within 24 hours of treatment, apoptosis in cultures of HEY ovarian cancer cells treated with neutralizing anti-STC1 monoclonal antibody was higher (17.3% apoptotic cells) than that in cultures treated with mouse IgG control cells (4.4%) (12.9% difference, 95% confidence interval = 11.6% to 14.2%). Conclusions STC1 protein may be involved in ovarian tumorigenesis.

Ovarian cancer survival and treatment have improved minimally in the past 20years. Novel treatment strategies are needed to combat this disease. This study investigates the effects of chemical inhibition of the CBFβ/RUNX protein-protein interaction on ovarian cancer cell lines. Ovarian cancer cell lines were treated with CBFβ/RUNX inhibitors, and the effects on proliferation, DNA replication, wound healing, and anchorage-independent growth were measured. RNA-Seq was performed on compound-treated cells to identify differentially expressed genes. Genes altered by compound treatment were targeted with siRNAs, and effects on DNA replication and wound healing were measured. Chemical inhibition of the CBFβ/RUNX interaction decreases ovarian cancer cell proliferation. Inhibitor treatment leads to an S-phase cell cycle delay, as indicated by an increased percentage of cells in S-phase, and a decreased DNA replication rate. Inhibitor treatment also reduces wound healing and anchorage-indepen...

Background-Hec1 (Highly-Expressed-in-Cancer) has recently been shown to play an important role in the proper segregation of chromosomes during mitosis. Recently, an Adenovirus delivery system carrying RNA interference (RNAi) of Hec1 has been reported in a cervical adenocarcinoma model. Adenoviral delivery systems, however, have the main limitation of poor viral infectivity due to lack of the native receptor, Coxsackie-Adenovirus Receptor (CAR), on the surface of tumor cells. We hypothesize that the viral infectivity of the Adenovirus vector would be enhanced via a CARindependent pathway by altering the targeting tropism, thus increasing the knockdown effect of Hec1 expression in ovarian carcinoma cells. Methods-Two Adenoviruses (Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3), along with a negative control (Ad-siRNA-GAPDH.F5/3) were created using homologous recombination. HEY and SKOV3.ip1 cell lines were used to perform experiments. The following assays were then used to determine RNAi knockdown efficiency: 1.Quantitative PCR (QPCR), 2.Western blot, 3.MTS Assay, 4.Annexin V-FITC FACS, 5.Crystal violet staining. In all experiments a negative control served as a baseline measure. Results-QPCR demonstrated a 2 log viral infectivity enhancement with Ad-siRNA-Hec1.F5/3 over Ad-siRNA-Hec1. QPCR at 72 hours revealed mRNA knockdown induced by Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3 in SKOV3.ip1 and HEY cells, respectively (71%/60%, and 32%/78% mRNA knockdown compared to negative control). Western blot revealed translational inhibition induced by both Hec1 Ads with the least knockdown seen with Ad-siRNA-GAPDH.F5/3. FACS analysis revealed increased Annexin V positivity in RNAi infected cells, suggesting a higher rate of apoptosis. MTS assay indicated increased cell death 8 days post-infection with Ad-siRNA-Hec1 and Ad-siRNA-Hec1.F5/3 in SKOV3.ip1 and HEY cell lines, respectively (75% vs. 35% and 43% vs.

The control of cell death is a biological process essential for proper development, and for preventing devastating pathologies like cancer and neurodegeneration. On the other hand, autophagy regulation is essential for protein and organelle degradation, and its dysfunction is associated with overlapping pathologies like cancer and neurodegeneration, but also for microbial infection and aging. In the present report we show that two evolutionarily unrelated receptors-Neurokinin 1 Receptor (NK 1 R,) a G-protein coupled receptor, and Insulin-like Growth Factor 1 Receptor (IGF1R), a tyrosine kinase receptor-both induce non-apoptotic cell death with autophagic features and requiring the activity of the autophagic core machinery proteins PI3K-III, Beclin-1 and Atg7. Remarkably, this form of cell death occurs in apoptosiscompetent cells. The signal transduction pathways engaged by these receptors both converged on the activation of the nuclear receptor NR4A1, which has previously been shown to play a critical role in some paradigms of apoptosis and in NK 1 R-induced cell death. The activity of NR4A1 was necessary for IGF1R-induced cell death, as well as for a canonical model of cell death by autophagy induced by the presence of a pan-caspase inhibitor, suggesting that NR4A1 is a general modulator of this kind of cell death. During cell death by autophagy, NR4A1 was transcriptionally competent, even though a fraction of it was present in the cytoplasm. Interestingly, NR4A1 interacts with the tumor suppressor p53 but not with Beclin-1 complex. Therefore the mechanism to promote cell death by autophagy might involve regulation of gene expression, as well as protein interactions. Understanding the molecular basis of autophagy and cell death mediation by NR4A1, should provide novel insights and targets for therapeutic intervention.

EVI1 (ecotropic viral integration site 1) is one of the most aggressive oncogenes associated with myeloid leukemia. We investigated DNA copy number aberrations in human hepatocellular carcinoma (HCC) cell lines using a high-density oligonucleotide microarray. We found that a novel amplification at the chromosomal region 3q26 occurs in the HCC cell line JHH-1, and that MECOM (MDS1 and EVI1 complex locus), which lies within the 3q26 region, was amplified. Quantitative PCR analysis of the three transcripts transcribed from MECOM indicated that only EVI1, but not the fusion transcript MDS1-EVI1 or MDS1, was overexpressed in JHH-1 cells and was significantly upregulated in 22 (61%) of 36 primary HCC tumors when compared with their non-tumorous counterparts. A copy number gain of EVI1 was observed in 24 (36%) of 66 primary HCC tumors. High EVI1 expression was significantly associated with larger tumor size and higher level of des-c-carboxy prothrombin, a tumor marker for HCC. Knockdown of EVI1 resulted in increased induction of the cyclin-dependent kinase inhibitor p15 INK4B by transforming growth factor (TGF)-b and decreased expression of c-Myc, cyclin D1, and phosphorylated Rb in TGF-b-treated cells. Consequently, knockdown of EVI1 led to reduced DNA synthesis and cell viability. Collectively, our results suggest that EVI1 is a probable target gene that acts as a driving force for the amplification at 3q26 in HCC and that the oncoprotein EVI1 antagonizes TGF-b-mediated growth inhibition of HCC cells.

Background A hallmark of epithelial ovarian cancer (EOC) metastasis is the process of spheroid formation, whereby tumour cells aggregate into 3D structures while in suspension in the peritoneal cavity. EOC spheroids are subjected to bioenergetic stress, thereby activating AMP-activated protein kinase (AMPK) signaling to enter a metabolically quiescent state, which can facilitate cell survival under nutrient-limiting conditions. Independently, we have also demonstrated that EOC spheroids induce autophagy, a process that degrades and recycles intracellular components to restore energy and metabolites. Herein, we sought to examine whether AMPK controls autophagy induction as a cell survival mechanism in EOC spheroids. Results We observed a co-ordinate increase in phosphorylated AMPK and the autophagy marker LC3-II during EOC spheroid formation. Reduced AMPK expression by siRNA-mediated knockdown of PRKAA1 and PRKAA2 blocked autophagic flux in EOC spheroids as visualized by fluorescence...

Crocin, a bioactive molecule of saffron, inhibited proliferation of both HCT116 wild-type and HCT116 p53(-/-) cell lines at a concentration of 10 mM. Flow cytometric analysis of cell cycle distribution revealed that there was an accumulation of HCT116 wild-type cells in G1 (55.9%, 56.1%) compared to the control (30.4%) after 24 and 48 h of crocin treatment, respectively. However, crocin induced only mild G2 arrest in HCT116 p53(-/-) after 24 h. Crocin induced inefficient autophagy in HCT116 p53(-/-) cells, where crocin induced the formation of LC3-II, which was combined with a decrease in the protein levels of Beclin 1 and Atg7 and no clear p62 degradation. Autophagosome formation was not detected in HCT116 p53(-/-) after crocin treatment predicting a nonfunctional autophagosome formation. There was a significant increase of p62 after treating the cells with Bafilomycin A1 (Baf) and crocin compared to crocin exposure alone. Annexin V staining showed that Baf-pretreatment enhanced th...

Phytochemicals derived from dietary sources and natural products have gained significant attention in the scientific community due to their ability to modulate various pharmacological and biological activities. Understanding the molecular mechanisms by which natural products protect against various diseases including cancer will provide the basis for both clinical use and further chemical modification to develop targeted therapy. Autophagy, an evolutionarily conserved self-digestion process that employs lysosomal-mediated enzymatic degradation has a functional role in a wide range of pathological disorders, and has attracted oncology scientists over the past two decades. Studies employing natural products have shown that induction of autophagy may be either cytoprotective or cytotoxic governed by different molecular pathways. In this review, we summarize four major phytochemicals namely phenethyl isothiocyanate, capsaicin, withaferin A, genistein and their association with autophagy...

Reduced autophagy may be associated with normal and pathological aging. Here we report a link between autophagy and Werner protein (WRNp), mutated in Werner syndrome, the human premature aging Werner syndrome (WS). WRN mutant fibroblast AG11395 and AG05229 respond weakly to starvation induced autophagy compared to normal cells. While the fusion of phagosomes with lysosome is normal, WS cells contain fewer autophagy vacuoles. Cellular starvation autophagy in WS cells is restored after transfection with full length WRN. Further, siRNA mediated silencing of WRN in the normal fibroblast cell line WI-38 results in decreased autophagy and altered expression of autophagy related proteins. Thus, our observations suggest that WRN may have a role in controlling autophagy and hereby cellular maintenance.

Nucleus accumbens-1 (NAC1), a nuclear factor belonging to the BTB/POZ gene family, is known to have important roles in proliferation and growth of tumor cells and in chemotherapy resistance. Yet, the mechanisms underlying how NAC1 contributes to drug resistance remain largely unclear. We report here that autophagy was involved in NAC1-mediated resistance to cisplatin, a commonly used chemotherapeutic drug in the treatment of ovarian cancer. We found that treatment with cisplatin caused an activation of ...

Autophagy is a conserved mechanism responsible for the continuous clearance of unnecessary organelles or misfolded proteins in lysosomes. Three types of autophagy have been reported in the difference of substrate delivery to lysosome: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Among these types, CMA is a unique autophagy system that selectively degrades substrates detected by heat shock cognate protein 70 (HSC70). Recently, autophagic cell death has been reported to be involved in neuronal death following brain ischemia; however, the contribution of CMA to neuronal death/survival after ischemic stress has not been addressed. In the present study, we determined whether quantitative alterations in LAMP-2A, which is the key molecule in CMA, would modulate neuronal cell survival under hypoxic conditions. Incubation of Neuro2A cells in a hypoxic chamber (1% O(2), 5% CO(2)) increased the level of LAMP-2A and induced accumulation of LAMP-2A-positive lysosomes in the perinuclear area, which is a hallmark of CMA activation. The activation of CMA in response to hypoxia was also confirmed by the GAPDH-HaloTag CMA indicator system at the single cell level. Next, we asked whether CMA was involved in cell survival during hypoxia. Blocking LAMP-2A expression with siRNA increased the level of cleaved caspase-3 and the number of propidium iodide-positive cells after hypoxic stress regardless of whether macroautophagy could occur, whereas the administration of mycophenolic acid, a potent CMA activator, rescued hypoxia-mediated cell death. Finally, we asked whether CMA was activated in the neurons after middle cerebral artery occlusion in vivo. The expression of LAMP-2A was significantly increased in the ischemic hemisphere seven days after brain ischemia. These results indicate that CMA is activated during hypoxia and contributes to the survival of cells under these conditions.

Indole-3-carbinol (I3C) is a broadly targeted phytochemical shown to prevent carcinogenesis in animal studies and to suppress the proliferation of cancer cells of human breast, colon, prostate, and endometrium. Here we demonstrate that OSU-A9, an I3C derivative with improved anticancer potency, induces cytotoxicity in acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells were less sensitive to OSU-A9 than leukemia cells. OSU-A9 induces caspase activation, PARP cleavage, and autophagy but not autophagic cell death. Interestingly, pretreatment of AML cell lines and primary AML cells with N-acetylcysteine or glutathione rescues them from apoptosis (and concomitant PARP cleavage) and Akt hypophosphorylation, implicating a key role of reactive oxygen species (ROS) in OSU-A9-related cytotoxicity. Importantly, the anticancer utility of OSU-A9 is extended in vivo as it, administered intraperitoneally, suppresses the growth of THP-1 xenograft tumors in athymic nude mice without obvious toxicity. This study shows that ROS-mediated apoptosis contributes to the anticancer activity of OSU-A9 in AML cell lines and primary AML cells, and thus should be considered in the future assessment of its translational value in AML therapy.

Reduced autophagy may be associated with normal and pathological aging. Here we report a link between autophagy and Werner protein (WRNp), mutated in Werner syndrome, the human premature aging Werner syndrome (WS). WRN mutant fibroblast AG11395 and AG05229 respond weakly to starvation induced autophagy compared to normal cells. While the fusion of phagosomes with lysosome is normal, WS cells contain fewer autophagy vacuoles. Cellular starvation autophagy in WS cells is restored after transfection with full length WRN. Further, siRNA mediated silencing of WRN in the normal fibroblast cell line WI-38 results in decreased autophagy and altered expression of autophagy related proteins. Thus, our observations suggest that WRN may have a role in controlling autophagy and hereby cellular maintenance.

Calcitriol or 1,25 dihydroxy vitamin D3, the hormonally active form of vitamin D, as well as vitamin D analogs, have been shown to increase sensitivity to ionizing radiation in breast tumor cells. The current studies indicate that the combination of 1,25 dihydroxy vitamin D3 with radiation appears to kill p53 wild type, estrogen receptor positive ZR-75-1 breast tumor cells through autophagy. Minimal apoptosis was observed based on cell morphology by DAPI and TUNEL staining, Annexin/PI analysis, Caspase-3 and PARP cleavage as well as cell cycle analysis. Induction of autophagy was indicated by increased acridine orange staining, RFP-LC3 redistribution and detection of autophagic vesicles by electron microscopy, while autophagic flux was monitored based on p62 degradation. The autophagy inhibitors, chloroquine and Bafilomycin A1, as well as genetic suppression of the autophagic signaling proteins Atg5 or Atg 7 attenuated the impact of the combination treatment of 1,25 D3 with radiation. In contrast to autophagy mediating the effects of the combination treatment, the autophagy induced by radiation alone was apparently cytoprotective in that either pharmacological or genetic inhibition increased sensitivity to radiation. These studies support the potential utility of vitamin D for improving the impact of radiation for breast cancer therapy, support the feasibility of combining chloroquine with radiation for the treatment of breast cancer and demonstrate the existence of an "autophagic switch" from

There is an unmet need for novel metal-based chemotherapeutics with alternative modes of action compared to clinical agents such as cisplatin and metallo-bleomycin. Recent attention in this field has focused on designing intracellular ROS-mediators as powerful cytotoxins of human cancers and identifying potentially unique toxic mechanisms underpinning their utility. Herein, we report the developmental di-manganese(II) therapeutic [Mn2(μ-oda)(phen)4(H2O)2][Mn2(μ-oda)(phen)4(oda)2]·4H2O (Mn-Oda) induces autophagy-promoted apoptosis in human ovarian cancer cells (SKOV3). The complex was initially identified to intercalate DNA by topoisomerase I unwinding and circular dichroism spectroscopy. Intracellular DNA damage, detected by γH2AX and the COMET assay, however, is not linked to direct Mn-Oda free radical generation, but is instead mediated through the promotion of intracellular reactive oxygen species (ROS) leading to autophagic vacuole formation and downstream nuclear degradation. T...

The TGF-β/Smad and the PI3K/AKT signaling pathways are important regulators of proliferation and apoptosis, and their alterations lead to cancer development. TGF-β acts as a tumor suppressor in premalignant cells, but it is a tumor promoter for cancerous cells. Such dichotomous actions are dictated by different cellular contexts. Here, we have unveiled a PTEN-Smad3 regulatory loop that provides a new insight in the complex cross talk between TGF-β/Smad and PI3K/AKT signaling pathways. We demonstrate that TGF-β triggers apoptosis of wild-type polarized endometrial epithelial cells by a Smad3-dependent activation of PTEN transcription, which results in the inhibition of PI3K/AKT signaling pathway. We show that specific Smad3 knockdown or knockout reduces basal and TGF-β-induced PTEN expression in endometrial cells, resulting in a blockade of TGF-β-induced apoptosis and an enhancement of cell proliferation. Likewise Smad3 deletion, PTEN knockout prevents TGF-β-induced apoptosis and inc...

The TGF-/Smad and the PI3K/AKT signaling pathways are important regulators of proliferation and apoptosis, and their alterations lead to cancer development. TGF-β acts as a tumor suppressor in premalignant cells, but it is a tumor promoter for cancerous cells. Such dichotomous actions are dictated by different cellular contexts. Here, we have unveiled a PTEN-Smad3 regulatory loop that provides a new insight in the complex crosstalk between TGF-/Smad and PI3K/AKT signaling pathways. We demonstrate that TGF- triggers apoptosis of wild type polarized endometrial epithelial cells by a Smad3-dependent activation of PTEN transcription, which results in the inhibition of PI3K/AKT signaling pathway. We show that specific Smad3 knock-down or knockout reduces basal and TGF--induced PTEN expression in endometrial cells, resulting in a blockade of TGF--induced apoptosis and an enhancement of cell proliferation. Likewise Smad3 deletion, PTEN knockout prevents TGF--induced apoptosis and increases cell proliferation by increasing PI3K/AKT/mTOR signaling. In summary, our results demonstrate that Smad3-PTEN signaling axis determine cellular responses to TGF-

Diesel exhaust particles (DEP) are known to generate reactive oxygen species in the respiratory system, triggering cells to activate antioxidant defence mechanisms, such as Keap1-Nrf2 signalling and autophagy. The aim of this study was to investigate the relationship between the Keap1-Nrf2 signalling and autophagy pathways after DEP exposure. BEAS-2B cells were transfected with silencing RNA (siRNA) specific to Nrf2 and exposed to DEP. The relative levels of mRNA for Nrf2, NQO1, HO-1, LC3B, p62 and Atg5 were determined using RT-PCR, while the levels of LCB3, Nrf2, and p62 protein were determined using Western blotting. The autophagy inhibitor bafilomycin caused a significant decrease in the production of Nrf2, HO-1 and NQO1 compared to DEPs treatment, whereas the Nrf2 activator sulforaphane increased the LC3B (p = 0.020) levels. BEAS-2B cells exposed to DEP at a concentration of 50 μg/mL for 2 h showed a significant increase in the expression of LC3B (p = 0.001), p62 (p = 0.008), Nrf2 (p = 0.003), HO-1 (p = 0.001) and NQO1 (p = 0.015) genes compared to control. In siRNA-transfected cells, the LC3B (p < 0.001), p62 (p = 0.001) and Atg5 (p = 0.024) mRNA levels and the p62 and LC3II protein levels were decreased, indicating that Nrf2 modulated the expression of autophagy markers (R < 1). These results imply that, in bronchial cells exposed to DEP, the Nrf2 system positively regulates autophagy to maintain cellular homeostasis.

Although endogenous nucleus pulposus-derived mesenchymal stem cell- (NPMSC-) based regenerative medicine has provided promising repair strategy for intervertebral disc (IVD) degeneration, the hostile microenvironments in IVD, including oxidative stress, can negatively affect the survival and function of the NPMSCs and severely hinder the endogenous repair process. Therefore, it is of great importance to reveal the mechanisms of the endogenous repair failure caused by the adverse microenvironments in IVD. The aim of this study was to investigate the effect of oxidative stress on the rat NPMSCs and its underlying mechanism. Our results demonstrated that oxidative stress inhibited cell viability, induced apoptosis, and increased the production of reactive oxygen species (ROS) in NPMSCs. In addition, the results showed that the expression level of heme oxygenase-1 (HO-1) increased at an early stage but decreased at a late stage when NPMSCs were exposed to oxidative stress, and the oxida...

Ovarian cancer is a leading cause of cancer death in women in the United States. While the majority of ovarian cancers are serous, some rarer subtypes (i.e. clear cell) are often associated with endometriosis, a benign gynecological disease. Iron is rich in the cyst fluid of endometriosis-associated ovarian cancers and induces persistent oxidative stress. The role of iron, an essential nutrient involved in multiple cellular functions, in normal ovarian cell survival and ovarian cancer remains unclear. Iron, presented as ferric ammonium citrate (FAC), dramatically inhibits cell survival in ovarian cancer cell types associated with Ras mutations, while it is without effect in immortalized normal ovarian surface epithelial (T80) and endometriotic epithelial cells (lacking Ras mutations). Interestingly, FAC induced changes in cytoplasmic vacuolation concurrently with increases in LC3-II levels (an autophagy marker); these changes occurred in an ATG5/ATG7-dependent, beclin-1/hVps34-indep...

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP)-1,-2,-9 and-12 gene expression and MMP-2 and-9 activities in SKOV3 and HEY1B ovarian cancer cells. MMP-1,-2,-9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells.

LC3 and Beclin-1 as Markers of Autophagic Activity in Breast Cancer Autophagy is a catabolic pathway meaning "self-eating" that facilitates nutrient recycling from damaged and aged organelles and other impaired cellular components through lysosomal degradation. Regulation of this process has been associated with the development of cancer. It can play different roles at different tumors and developmental stages of tumors. In breast cancer, similarly, autophagy functions as a mechanism promoting survival or leading to death. Whereas, it is very important to define the role of autophagy as an effective treatment strategy in breast cancer cells. Therefore, in this review, the role of inhibited autophagy is discussed with specific RNAs targeting Beclin-1 and LC3 genes in breast cancer.

Objective. To determine whether Arsenic Trioxide may be an effective treatment of human ovarian carcinoma, we examined if it induced apoptosis in human ovarian cancer cell line. Methods. The human ovarian cell line SKOV3 was exposed to different concentrations of As 2 O 3 : 0 (control), 0.1 AM, 1 AM, 5 AM and 10 AM. The effect on cells proliferation and apoptotic parameters was examined. Results. The most effective inhibitory Arsenic Trioxide concentrations were 5 AM and 10 AM, causing growth inhibition of 79% and 83%, respectively. The maximum inhibitory effect of Arsenic Trioxide on cellular proliferation was seen after 48 h. No morphological or molecular features of apoptosis have been observed. At the same time, there were no typical changes of cellular necrosis. Conclusions. The exact mechanism by which Arsenic Trioxide inhibits the growth of ovarian cancer cells is probably not by apoptosis. However, since As 2 O 3 has been shown to inhibit ovarian cancer cellular growth, it should be further elucidated as a possible chemotherapeutic agent of ovarian cancer.

Insulin-like growth factor II (IGF-II) plays a pivotal role in fetal and cancer development by signaling through the IGF-I and insulin receptors and activating the estrogen signaling cascade. We previously showed that precursor IGF-II (proIGF-II, the predominant form expressed in cancer) and not mature IGF-II (mIGF-II) blocks resveratrol (RSV) (a phytoalexin/anticancer agent)-induced cell death in MCF-7 cells. We hypothesize that proIGF-II regulates antiapoptotic proteins and/or the mitochondria to inhibit RSV actions and promote cell survival. This study examines the effect of mIGF-II and proIGF-II on survivin expression and mitochondrial polarization in response to RSV. RSV inhibits survivin expression and stimulates mitochondrial depolarization, caspase 7 activation and cell death. These effects were completely blocked by the addition of proIGF-II. RSV treatment had no effect on transfected MCF-7 cells constitutively expressing proIGF-II, while IGF-II siRNA transfection decreased survivin levels. Our results provide new insights for the potential use of proIGF-II as target for new anticancer therapies.

Ultraviolet-B (UV-B) exposure to the skin causes photo-damage and acts as the primary etiological agent in photo-carcinogenesis. UV-B exposure induces photodamage in epidermal cells and is the major factor that challenges skin homeostasis. Autophagy allows fundamental adaptation of cells to metabolic needs and stresses. Cellular dysfunction is observed in aged tissues and in toxic insults to cells that undergo stress. Conversely, promising anti-ageing strategies aimed at inhibiting the mTOR pathway has been found to significantly improve the ageing-related disorders. Recently, autophagy has been found to positively regulate skin homeostasis by enhancing DNA damage recognition. Here we investigated the Geno-protective roles of autophagy in UV-B exposed primary HDFs. We found that improving autophagy levels in HDFs regulates UV-B mediated cellular stress by decreasing the formation of DNA photo adducts, alleviates oxidative and ER stress response and by regulating the expression levels of cell cycle regulatory proteins P21 and P27. Autophagy also prevents HDFs from UV-B -induced nuclear damage as is evident from Tunnel assay and Acridine Orange/Ethidium Bromide co-staining. Salubrinal, (an eIf2α inhibitor) significantly decreases the DNA damage response in HDFs. P62 silenced HDFs show enhanced DNA damage response and disturbs the tumour suppressor axis PTEN/pAKT towards damage whereas ATG7 silenced HDFs reveal an unexpected consequence by decreasing the UV-B -induced DNA damage compared to UV-B treated HDFs. Together, our results suggest that autophagy is essential in protecting skin cells from UV-B radiation-induced photo-damage and holds great promise in devising it as a suitable therapeutic strategy against skin photo-damage.