Javad Alizadeh

Cellular commitment in the developing cerebellum

The mammalian cerebellum is located in the posterior cranial fossa and is critical for motor coordination and non-motor functions including cognitive and emotional processes. The anatomical structure of cerebellum is distinct with a three-layered cortex. During development, neurogenesis and fate decisions of cerebellar primordium cells are orchestrated through tightly controlled molecular events involving multiple genetic pathways. In this review, we will highlight the anatomical structure of human and mouse cerebellum, the cellular composition of developing cerebellum, and the underlying gene expression programs involved in cell fate commitments in the cerebellum. A critical evaluation of the cell death literature suggests that apoptosis occurs in ~5% of cerebellar cells, most shortly after mitosis. Apoptosis and cellular autophagy likely play significant roles in cerebellar development, we provide a comprehensive discussion of their role in cerebellar development and organization. We also address the possible function of unfolded protein response in regulation of cerebellar neurogenesis. We discuss recent advancements in understanding the epigenetic signature of cerebellar compartments and possible connections between DNA methylation, microRNAs and cerebellar neurodegeneration. Finally, we discuss genetic diseases associated with cerebellar dysfunction and their role in the aging cerebellum.

Hepatitis B and C virus-induced hepatitis: Apoptosis, autophagy, and unfolded protein response

AIM To investigate the co-incidence of apoptosis, autophagy, and unfolded protein response (UPR) in hepatitis B (HBV) and C (HCV) infected hepatocytes. METHODS We performed immunofluorescence confocal microscopy on 10 liver biopsies from HBV and HCV patients and tissue microarrays of HBV positive liver samples. We used specific antibodies for LC3β, cleaved caspase-3, BIP (GRP78), and XBP1 to detect autophagy, apoptosis and UPR, respectively. Anti-HCV NS3 and anti-HBs antibodies were also used to confirm infection. We performed triple blind counting of events to determine the co-incidence of autophagy (LC3β punctuate), apoptosis (cleaved caspase-3), and unfolded protein response (GRP78) with HBV and HCV infection in hepatocytes. All statistical analyses were performed using SPSS software for Windows (Version 16 SPSS Inc, Chicago, IL, United States). P-values < 0.05 were considered statistically significant. Statistical analyses were performed with Mann-Whitney test to compare incidence rates for autophagy, apoptosis, and UPR in HBV- and HCV-infected cells and adjacent non-infected cells. RESULTS Our results showed that infection of hepatocytes with either HBV and HCV induces significant increase (P < 0.001) in apoptosis (cleavage of caspase-3), autophagy (LC3β punctate), and UPR (increase in GRP78 expression) in the HCV- and HBV-infected cells, as compared to non-infected cells of the same biopsy sections. Our tissue microarray immunohistochemical expression analysis of LC3β in HBV(Neg) and HBV(Pos) revealed that majority of HBV-infected hepatocytes display strong positive staining for LC3β. Interestingly, although XBP splicing in HBV-infected cells was significantly higher (P < 0.05), our analyses show a slight increase of XBP splicing was in HCV-infected cells (P > 0.05). Furthermore, our evaluation of patients with HBV and HCV infection based on stage and grade of the liver diseases revealed no correlation between these pathological findings and induction of apoptosis, autophagy, and UPR. CONCLUSION The results of this study indicate that HCV and HBV infection activates apoptosis, autophagy and UPR, but slightly differently by each virus. Further studies are warranted to elucidate the interconnections between these pathways in relation to pathology of HCV and HBV in the liver tissue.

New frontiers in the treatment of colorectal cancer: Autophagy and the unfolded protein response as promising targets

ABSTRACT Colorectal cancer (CRC), despite numerous therapeutic and screening attempts, still remains a major life-threatening malignancy. CRC etiology entails both genetic and environmental factors. Macroautophagy/autophagy and the unfolded protein response (UPR) are fundamental mechanisms involved in the regulation of cellular responses to environmental and genetic stresses. Both pathways are interconnected and regulate cellular responses to apoptotic stimuli. In this review, we address the epidemiology and risk factors of CRC, including genetic mutations leading to the occurrence of the disease. Next, we discuss mutations of genes related to autophagy and the UPR in CRC. Then, we discuss how autophagy and the UPR are involved in the regulation of CRC and how they associate with obesity and inflammatory responses in CRC. Finally, we provide perspectives for the modulation of autophagy and the UPR as new therapeutic options for CRC treatment.

Apoptosis, autophagy and unfolded protein response pathways in Arbovirus replication and pathogenesis.

Arboviruses are pathogens that widely affect the health of people in different communities around the world. Recently, a few successful approaches toward production of effective vaccines against some of these pathogens have been developed, but treatment and prevention of the resulting diseases remain a major health and research concern. The arbovirus infection and replication processes are complex, and many factors are involved in their regulation. Apoptosis, autophagy and the unfolded protein response (UPR) are three mechanisms that are involved in pathogenesis of many viruses. In this review, we focus on the importance of these pathways in the arbovirus replication and infection processes. We provide a brief introduction on how apoptosis, autophagy and the UPR are initiated and regulated, and then discuss the involvement of these pathways in regulation of arbovirus pathogenesis.

Mevalonate Cascade Inhibition by Simvastatin Induces the Intrinsic Apoptosis Pathway via Depletion of Isoprenoids in Tumor Cells

The mevalonate (MEV) cascade is responsible for cholesterol biosynthesis and the formation of the intermediate metabolites geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) used in the prenylation of proteins. Here we show that the MEV cascade inhibitor simvastatin induced significant cell death in a wide range of human tumor cell lines, including glioblastoma, astrocytoma, neuroblastoma, lung adenocarcinoma, and breast cancer. Simvastatin induced apoptotic cell death via the intrinsic apoptotic pathway. In all cancer cell types tested, simvastatin-induced cell death was not rescued by cholesterol, but was dependent on GGPP- and FPP-depletion. We confirmed that simvastatin caused the translocation of the small Rho GTPases RhoA, Cdc42, and Rac1/2/3 from cell membranes to the cytosol in U251 (glioblastoma), A549 (lung adenocarcinoma) and MDA-MB-231(breast cancer). Simvastatin-induced Rho-GTP loading significantly increased in U251 cells which were reversed with MEV, FPP, GGPP. In contrast, simvastatin did not change Rho-GTP loading in A549 and MDA-MB-231. Inhibition of geranylgeranyltransferase I by GGTi-298, but not farnesyltransferase by FTi-277, induced significant cell death in U251, A549, and MDA-MB-231. These results indicate that MEV cascade inhibition by simvastatin induced the intrinsic apoptosis pathway via inhibition of Rho family prenylation and depletion of GGPP, in a variety of different human cancer cell lines.

Interconnection of Estrogen/Testosterone Metabolism and Mevalonate Pathway in Breast and Prostate Cancers

The metabolic steroid hormones, 17β stradiol (E2) and testosterone play key roles in several functions including carbohydrate, lipid and protein metabolism, cellular signaling, cell proliferation, and cancer promotion. Steroid hormones have long been characterized as cell proliferation and differentiation regulators and are closely related to the development of breast and prostate cancers. Moreover, cholesterol metabolism, mainly in adipose tissue, leads to the production of steroids and cytokines, thus increasing the risk of metabolic syndrome, obesity, and ER+ breast cancer in postmenopausal women. Recent studies also shown that testosterone and E2 increase the levels of key enzymes of the mevalonate pathway, leading to post-translational prenylation and farnesylation of numerous proteins in RAS signaling in several cancers, including breast and prostate cancers. There is accumulating evidence both clinically and experimentally suggesting that changes in the metabolism of cholesterol may also have an important role in carcinogenesis. In this regard, the cells treated with mevalonate in culture showed elevated proliferation. Therefore, investigation on cholesterol as a precursor of steroid hormones has confirmed the effects cholesterol metabolite on breast and prostate cancers. Indeed, recent evidence strongly suggests that the MVA pathway and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCOA) have a crucial regulatory role in cellular proliferation and transformation. Therefore, the use of mevalonate inhibitors decreases the production of several biologically active downstream products of the mevalonate pathway, including cholesterol. Although for approximately 20 years statins have been identified as anticancer agents, recent studies have sparked some controversy. Therefore, further investigation to evaluate mevalonate- dependent therapeutic agents per se and in combination with other agents is merited. The current review is an attempt to elucidate the role of cholesterol and E2/testosterone, as well as the mevalonate pathway and its inhibitors in breast and prostate tissues in normal and pathological states.

Autophagy modulates transforming growth factor beta 1 induced epithelial to mesenchymal transition in non-small cell lung cancer cells

Lung cancer is considered one of the most frequent causes of cancer-related death worldwide and Non-Small Cell Lung Cancer (NSCLC) accounts for 80% of all lung cancer cases. Autophagy is a cellular process responsible for the recycling of damaged organelles and protein aggregates. Transforming growth factor beta-1 (TGFβ1) is involved in Epithelial to Mesenchymal Transition (EMT) and autophagy induction in different cancer models and plays an important role in the pathogenesis of NSCLC. It is not clear how autophagy can regulate EMT in NSCLC cells. In the present study, we have investigated the regulatory role of autophagy in EMT induction in NSCLC and show that TGFβ1 can simultaneously induce both autophagy and EMT in the NSCL lines A549 and H1975. Upon chemical inhibition of autophagy using Bafilomycin-A1, the expression of the mesenchymal marker vimentin and N-cadherin was reduced. Immunoblotting and immunocytochemistry (ICC) showed that the mesenchymal marker vimentin was significantly downregulated upon TGFβ1 treatment in ATG7 knockdown cells when compared to corresponding cells treated with scramble shRNA (negative control), while E-cadherin was unchanged. Furthermore, autophagy inhibition (Bafilomycin A1 and ATG7 knockdown) decreased two important mesenchymal functions, migration and contraction, of NSCLC cells upon TGFβ1 treatment. This study identified a crucial role of autophagy as a potential positive regulator of TGFβ1-induced EMT in NSCLC cells and identifies inhibitors of autophagy as promising new drugs in antagonizing the role of EMT inducers, like TGFβ1, in the clinical progression of NSCLC.

Autophagy and the Unfolded Protein Response Promote Pro-fibrotic Effects of TGFβ1 in Human Lung Fibroblasts

Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease in adults with limited treatment options. Autophagy and the unfolded protein response (UPR), fundamental processes induced by cell stress, are dysregulated in lung fibroblasts and epithelial cells from humans with IPF. Human primary cultured lung parenchymal and airway fibroblasts from non-IPF and IPF donors were stimulated with transforming growth factor-β1 (TGF-β1) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3βII and Atg5-12), UPR (BIP, IRE1α, and cleaved XBP1), and fibrosis (collagen 1α2 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3βII) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-β1-induced collagen 1α2 and fibronectin protein production was significantly higher in IPF lung fibroblasts compared with lung and airway fibroblasts from non-IPF donors. TGF-β1 induced the accumulation of LC3βII in parallel with collagen 1α2 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-β1-induced collagen and fibronectin biosynthesis was significantly reduced by inhibiting autophagy flux in fibroblasts from the lungs of non-IPF and IPF donors. Conversely, only in lung fibroblasts from IPF donors did TGF-β1 induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-β1-induced collagen 1α2 and fibronectin biosynthesis in IPF lung fibroblasts but not those from non-IPF donors. The IRE1 arm of the UPR response is uniquely induced by TGF-β1 in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these cells.

Simultaneous Detection of Autophagy and Epithelial to Mesenchymal Transition in the Non-small Cell Lung Cancer Cells

Autophagy is increasingly identified as a central player in many cellular activities from cell proliferation to cell division, migration, and differentiation. However, it is also considered as a double-edged sword in cancer biology which either promotes oncogenesis/invasion or sensitizes the tumor cells to chemotherapy induced apoptosis. Recent investigations have provided direct evidence for regulation of cellular phenotype via autophagy pathway. One of the most important types of phenotype conversion is Epithelial-Mesenchymal-Transition (EMT), resulting in alteration of epithelial cell properties to a more mesenchymal form. In the current chapter, we provide a method which is established and being used in our laboratory for detection of autophagy and EMT in lung epithelial cells and show the involvement of autophagy in modulation of cellular phenotype.

Autophagy modulates temozolomide-induced cell death in alveolar Rhabdomyosarcoma cells

Rhabdomyosarcoma (RMS) is a muscle-derived tumor. In both pre-clinical and clinical studies Temozolomide (TMZ) has been recently tested against RMS; however, the precise mechanism of action of TMZ in RMS remains unclear. Here we demonstrate that TMZ decreases the cell viability of the RH30 RMS and C2C12 cell line, where cells display evidence of mitochondrial outer membrane permeability. Interestingly, the C2C12 mouse myoblast line was relatively more resistant to TMZ-induced apoptosis. Moreover, we observed that TMZ activated biochemical and morphological markers of autophagy in both cell lines. Autophagy inhibition in both RH30 and C2C12 cells significantly increased TMZ-induced cell death. In RH30 cells, TMZ increased Mcl-1 and Bax protein expression compared to corresponding time match controls while in C2C12 Mcl-1, Bcl-2, Bcl-XL, and Bax protein expression were not changed. Baf-A1 co-treatment with TMZ significantly decrease Mcl-1 expression compared to TMZ while increase Bax expression in C2C12 cells (Bcl2 and Bcl-XL do not significantly change in Baf-A1/TMZ co-treatment). Using a three-dimensional (3D) C2C12 and RH30 culture model we demonstrated that TMZ is significantly more toxic in RH30 cells (live/dead assay). Additionally, we have observed in our 3D culture model that TMZ induced both apoptosis (cleavage of PARP) and autophagy (LC3-puncta and localization of LC3/p62). Therefore, our data demonstrate that TMZ induces simultaneous autophagy and apoptosis in both RH30 and C2C12 cells in 2D and 3D culture model, where RH30 cells are more sensitive to TMZ-induced death. Furthermore, autophagy serves to protect RH30 cells from TMZ-induced death.