Combining high-throughput screening of caspase activity with anti-apoptosis genes for development of robust CHO production cell lines

A set of anti-apoptotic genes were over-expressed, either singly or in combination, in an effort to develop robust Chinese Hamster Ovary host cell lines suitable for manufacturing biotherapeutics. High-throughput screening of caspase 3/7 activity enabled a rapid selection of transfectants with reduced caspase activity relative to the host cell line. Transfectants with reduced caspase 3/7 activity were then tested for improved integrated viable cell count (IVCC), a function of peak viable cell density and longevity. The maximal level of improvement in IVCC could be achieved by over-expression of either single anti-apoptotic genes, e.g., Bcl-2Δ (a mutated variant of Bcl-2) or Bcl-XL, or a combination of two or three anti-apoptotic genes, e.g., E1B-19K, Aven, and XIAPΔ. These cell lines yielded higher transient antibody production and a greater number of stable clones with high antibody yields. In a 5 L fed-batch bioreactor system, BΔ31-1, a stable clone expressing Bcl-2Δ, had a product titer that was 180% as compared to an optimal clone (Con-1) from the control cell line. Although lactate accumulated to more than 5 g/L in the control culture, its concentration was reduced in the anti-apoptotic BΔ31-1 cultures to below 1 g/L, confirming our earlier findings that cells over-expressing anti-apoptotic genes consume the lactate that would otherwise accumulate as a by-product in the culture medium. To the best of our knowledge, this is the first study to use the high throughput caspase screening method to identify CHO host cell lines with superior anti-apoptotic characteristics.     

Effect of Bcl‐xL overexpression on lactate metabolism in chinese hamster ovary cells producing antibody

Previously, overexpression of anti‐apoptotic proteins, such as E1B‐19K and Aven, was reported to alter lactate metabolism of CHO cells in culture. To investigate the effect of Bcl‐x_L, a well‐known anti‐apoptotic protein, on lactate metabolism of recombinant CHO (rCHO) cells, two antibody‐producing rCHO cell lines with regulated Bcl‐x_L overexpression (CS13*‐0.02‐off‐Bcl‐x_L and CS13*‐1.00‐off‐Bcl‐x_L) were established using the Tet‐off system. When cells were cultivated without Bcl‐x_L overexpression, the specific lactate production rate (q_Lac) of CS13*‐0.02‐off‐Bcl‐x_L and CS13*‐1.00‐off‐Bcl‐x_L were 7.32 ± 0.37 and 6.78 ± 0.56 pmol/cell/day, respectively. Bcl‐x_L overexpression, in the absence of doxycycline, did not affect the q_Lac of either cell line, though it enhanced the viability during cultures. Furthermore, activities of the enzymes related to glucose and lactate metabolism, such as hexokinase, glucose‐6‐phosphate dehydrogenase, lactate dehydrogenases, and alanine aminotransferase, were not affected by Bcl‐x_L overexpression either. Taken together, Bcl‐x_L overexpression showed no significant effect on the lactate metabolism of rCHO cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1594–1598, 2013     

Combining caspase and mitochondrial dysfunction inhibitors of apoptosis to limit cell death in mammalian cell cultures

Apoptosis is now recognized as a significant problem in mammalian cell culture. Therefore, in this study, a single gene and multigene approach to inhibit apoptosis has been examined. Stable Chinese hamster ovary (CHO) cell lines were generated to overexpress different single, dual, and triple combinations of three apoptosis inhibitor genes. Two upstream inhibitors involved in the mitochondrial pathway, Bcl-X(L) and Aven, were expressed in addition to a downstream inhibitor of caspases. The caspase inhibitor, a variant of XIAP containing only the caspase inhibitory BIR domains (XIAP-BIRs), has been shown previously to enhance viabilities in mammalian cultures. Stable clonal cell lines were generated and tested for three apoptotic insults: Sindbis virus infection, the chemical reagent etoposide, and spent medium. For all single gene experiments, the Bcl-X(L)-containing cell lines provided superior protection to either the Aven- or XIAP-BIRs-containing cell lines following initial exposure to the insults. However, the cell lines expressing two or more anti-apoptosis proteins were more effective at inhibiting cell death than those expressing just one anti-apoptosis gene. The cell lines overexpressing Bcl-X(L) in combination with XIAP-BIRs were especially effective in delaying cell death for all three apoptotic insults. Expression of all three anti-apoptosis genes in concert was only slightly more effective than using Bcl-X(L) and XIAP-BIRs for some insults. During exposure to spent medium, CHO-BIRS + Aven + BclX(L) was the best inhibitor of apoptosis (IAP) initially, whereas CHO-BIRs + BclX(L) was particularly effective at later times of the experiment. In conclusion, the utilization of a mitochondrial dysfunction inhibitor used in combination with a caspase inhibitor was more effective in thwarting the progression of apoptosis than either inhibitor expressed individually. Thus, the concurrent expression of multiple apoptosis inhibitors may be the most effective strategy to increase survival of mammalian cells in culture.     

Modulation of caspases and their non‐apoptotic functions by Legionella pneumophila

Legionella pneumophila has become a model system to decipher the non‐apoptotic functions of caspases and their role in immunity. In permissive cells, the L. pneumophila‐containing vacuole evades endosomal traffic and is remodelled by the endoplasmic reticulum. Evasion of the endosomes is mediated by the Dot/Icm type IV secretion system. Upon L. pneumophila infection of genetically restrictive cells such as wild‐type (WT) C57Bl/6J murine macrophages, flagellin is sensed by the NOD‐like receptor Nlrc4 leading to caspase‐1 activation by the inflammasome complex. Then, caspase‐7 is activated downstream of the Nlrc4 inflammasome, promoting non‐apoptotic functions such as L. pneumophila‐containing phagosome maturation and bacterial degradation. Interestingly, caspase‐3 is activated in permissive cells during early stages of infection. However, caspase‐3 activation does not lead to apoptosis until late stages of infection because it is associated with potent Dot/Icm‐mediated anti‐apoptotic stimuli that render the infected cells resistant to external apoptotic inducers. Therefore, the role of caspase‐1 and non‐apoptotic functions of executioner caspases are temporally and spatially modulated during infection by L. pneumophila, which determine permissiveness to intracellular bacterial proliferation. This review will examine the novel activation pathways of caspases by L. pneumophila and discuss their role in genetic restriction and permissiveness to infection.     

Induction of caspase 3 activation by multiple Legionella pneumophila Dot/Icm substrates

The intracellular pathogen Legionella pneumophila is able to strike a balance between the death and survival of the host cell during infection. Despite the presence of high level of active caspase 3, the executioner caspase of apoptotic cell death, infected permissive macrophages are markedly resistant to exogenous apoptotic stimuli. Several bacterial molecules capable of promoting the cell survival pathways have been identified, but proteins involved in the activation of caspase 3 remain unknown. To study the mechanism of L. pneumophila‐mediated caspase 3 activation, we tested all known Dot/Icm substrates for their ability to activate caspase 3. Five effectors capable of causing caspase 3 activation upon transient expression were identified. Among these, by using its ability to activate caspase 3 by inducing the release of cytochrome c from the mitochondria, we demonstrated that VipD is a phospholipase A2, which hydrolyses phosphatidylethanolamine (PE) and phosphocholine (PC) on the mitochondrial membrane in a manner that appears to require host cofactor(s). The lipase activity leads to the production of free fatty acids and 2‐lysophospholipids, which destabilize the mitochondrial membrane and may contribute to the release of cytochrome c and the subsequent caspase 3 activation. Furthermore, we found that whereas it is not detectably defectively in caspase 3 activation in permissive cells, amutant lacking all of these five genes is less potent in inducing apoptosis in dendritic cells. Our results reveal that activation of host cell death pathways by L. pneumophila is a result of the effects of multiple bacterial proteins with diverse biochemical functions.     

Activity of different anthracycline formulations in hormone-refractory prostate cancer cell lines: role of Golgi apparatus

The efficacy of therapy for hormone‐refractory prostate cancer (HRPC) is still unsatisfactory and new agents and therapeutic modalities are needed. The aims of the present work were to examine the in vitro activity and mechanisms of action of doxorubicin (DX), pegylated liposomal DX (PLDX), and non‐pegylated liposomal DX (NPLDX) in DU145 and taxane‐resistant DU145‐R HRPC cell lines. Drug activity and incorporation, apoptosis, and expression of cell death‐related markers were evaluated by SRB test, cytofluorimetric assays, and Western blot, respectively. Among the different DX formulations, NPLDX showed the highest cytotoxic activity in both cell lines, with more than 50% of apoptotic cells at only 1/10 of the plasma peak concentration after 72 h exposure. Anthracyclines, in particular NPLDX, were highly concentrated in the Golgi apparatus. Moreover, a significant increase was observed in the expression of CD95 receptor, GD3 ganglioside and, caspase‐2 and ‐8 active forms in both cell lines followed by caspase‐3 activation and mitochondrial membrane depolarization. The Golgi apparatus, probably acting as a stress sensor, intensified the conventional apoptotic mechanism induced by anthracyclines. Our data support the hypothesis that organelle‐dependent initiation of cell death other than that induced by mitochondria and nucleus is a research area worthy of pursuing and suggest that the Golgi apparatus could be an ideal target for anti‐cancer therapy. Of note, the activity of NLPDX in taxane‐resistant DU145‐R cells warrants further evaluation as second‐line treatment of advanced HRPC after taxane failure. J. Cell. Physiol. 226: 3035–3042, 2011. © 2011 Wiley‐Liss, Inc.     

Inhibition of the PI3K/AKT pathway potentiates cytotoxicity of EGFR kinase inhibitors in triple‐negative breast cancer cells

Triple‐negative breast cancers (TNBCs) are known to be intrinsically resistant to inhibitors for epidermal growth factor receptor (EGFR). Until now, clinical trials for TNBCs using EGFR inhibitors (EGFRis) as single agents have yielded disappointing results. Here, we report that combinatorial treatment using EGFRis, such as gefitinib or erlotinib, with PI3K/AKT pathway inhibitors (PI3K/AKTis) demonstrated a synergistic, anti‐proliferative effect in cell lines of the basal‐like (BL) subtype, a subtype of TNBC. Western blot analysis revealed that the gefitinib/PI‐103 combination significantly reduced the level of both phospho‐AKT and phospho‐ERK in two susceptible BL subtype cell lines, SUM149PT and MDA‐MB‐468, whereas it had little or no effect on the level of phospho‐ERK in two non‐susceptible cell lines (HS578T and MDA‐MB‐231) of mesenchymal stem‐like (MSL) TNBC subtype. The gefitinib/PI‐103 combination also significantly induced caspase‐3/7‐mediated PARP cleavage and reduced two anti‐apoptotic proteins, XIAP and Bcl‐2 in the susceptible cell lines. In addition, the level of myeloid cell leukemia 1 (Mcl‐1) protein was markedly decreased by gefitinib/PI‐103 combination in the BL TNBC cells, but showed no significant change by this combination in MSL subtype cells. These results suggest that pharmacological inhibition of EGFR used in combination of PI3K/AKTis is a potential therapeutic approach to treat a subtype of TNBCs.     

Aven, a novel inhibitor of caspase activation, binds Bcl-xL and Apaf-1

Bcl-x(L), an antiapoptotic Bcl-2 family member, is postulated to function at multiple stages in the cell death pathway. The possibility that Bcl-x(L) inhibits cell death at a late (postmitochondrial) step in the death pathway is supported by this report of a novel apoptosis inhibitor, Aven, which binds to both Bcl-x(L) and the caspase regulator, Apaf-1. Identified in a yeast two-hybrid screen, Aven is broadly expressed and is conserved in other mammalian species. Only those mutants of Bcl-x(L)that retain their antiapoptotic activity are capable of binding Aven. Aven interferes with the ability of Apaf-1 to self-associate, suggesting that Aven impairs Apaf-1-mediated activation of caspases. Consistent with this idea, Aven inhibited the proteolytic activation of caspases in a cell-free extract and suppressed apoptosis induced by Apaf-1 plus caspase-9. Thus, Aven represents a new class of cell death regulator.     

Use of an anti‐apoptotic CHO cell line for transient gene expression

Transient gene expression in mammalian cells allows for rapid production of recombinant proteins for research and preclinical studies. Here, we describe the development of a polyethylenimine (PEI) transient transfection system using an anti‐apoptotic host cell line. The host cell line, referred to as the Double Knockout (DKO), was generated by deleting two pro‐apoptotic factors, Bax and Bak, in a CHO‐K1 cell line using zinc finger nuclease mediated gene disruption. Optimized DNA and PEI volumes for DKO transfections were 50% and 30% lower than CHO‐K1, respectively. During transfection DKO cells produced relatively high levels of lactate, but this was mitigated by a temperature shift to 31°C which further enhanced productivity. DKO cells expressed ～3‐ to 4‐fold higher antibody titers than CHO‐K1 cells. As evidence of their anti‐apoptotic properties post‐transfection, DKO cells maintained higher viability and had reduced levels of active caspase‐3 compared to CHO‐K1 cells. Nuclear plasmid DNA copy numbers and message levels were significantly elevated in DKO cells. Although DNA uptake levels, as early as 40 min post‐transfection, were higher in DKO cells this was not due to differences in cell surface heparan sulfate (HS) or initial endocytosis mechanism as both cell types utilized caveolae‐ and clathrin‐mediated endocytosis to internalize DNA:PEI complexes. These results suggest that the increased transfection efficiency and titers from DKO cells are attributed to their resistance to transfection‐induced apoptosis and not differences in endocytosis mechanism. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1050–1058, 2013     

Increased anaerobic metabolism is a distinctive signature in a colorectal cancer cellular model of resistance to antiepidermal growth factor receptor antibody

Cetuximab is a chimeric antibody approved for the treatment of metastatic colorectal cancer that selectively targets epidermal growth factor receptor (EGFR) signaling. Treatment efficacy with this drug is often impaired by acquired resistance and poor information has been accumulated on the mechanisms underlying such a phenomenon. By taking advantage of a syngenic cellular system of sensitivity and acquired resistance to anti‐EGFR therapy in the colorectal carcinoma GEO cell line, we profiled protein expression differences between Cetuximab‐sensitive and ‐resistant cells. Combined 2D DIGE and MS analyses revealed a main proteomic signature resulting from selective deregulation of various metabolic enzymes, including glucose‐6‐phosphate dehydrogenase, transketolase, lactate dehydrogenase B, and pyruvate dehydrogenase E1, which was also confirmed by Western blotting experiments. Lactate dehydrogenase B downregulation has been already related to an increased anaerobic utilization of glucose by tumor cells; accordingly, we verified that Cetuximab‐resistant cells have a significantly higher production of lactate. Resistant cells also showed decreased nicotinamide adenine dinucleotide phosphate (NADPH) levels. Observed protein deregulations were not related to functional alterations of the hypoxia‐inducible factor 1‐associated pathways. Our data demonstrate that increased anaerobic metabolism is a prominent feature observed in the GEO syngenic model of acquired resistance to anti‐EGFR therapy in colorectal cancer.     

Lactate Formation in Primary and Metastatic Colon Cancer Cells at Hypoxia and Normoxia

High glucose consumption and lactate synthesis in aerobic glycolysis are a hallmark of cancer cells. They can form lactate also in glutaminolysis, but it is not clear how oxygen availability affects this process. We studied lactate synthesis at various oxygen levels in human primary (SW480) and metastatic (SW620) colon cancer cells cultured with L‐Ser and/or L‐Asp. Glucose and lactate levels were determined colorimetrically, amino acids by HPLC, expression of AST1‐mRNA and AST2‐mRNA by RT‐PCR. In both lines glucose consumption and lactate synthesis were higher at 10% than at 1% oxygen, and lactate/glucose ratio was increased above 2.0 by L‐Asp. AST1‐mRNA expression was independent on oxygen and cell line, but AST2‐mRNA was lower at hypoxia in SW480. We conclude that, in both cell lines at 1% hypoxia, lactate is formed mainly from glucose but at 10% normoxia also from L‐Asp. At 10% normoxia, lactate synthesis is more pronounced in primary than metastatic colon cancer cells.     

Protein-free fed-batch culture of non-GS NS0 cell lines for production of recombinant antibodies

Presented is a novel antibody production platform based on the fed-batch culture of recombinant, NS0-derived cell lines. A standardized fed-batch cell culture process was developed for five non-GS NS0 cell lines using enriched and optimized protein-free, cholesterol-free, and chemically defined basal and feed media. The process performed reproducibly and scaled faithfully from the 2-L to the 100-L bioreactor scale achieving a volumetric productivity of > 120 mg/L per day. Fed-batch cultures for all five cell lines exhibited significant lactate consumption when the cells entered the stationary or death phase. Peak and final lactate concentrations were low relative to a previously developed fed-batch process (FBP). Such low lactate production and high lactate consumption rates were unanticipated considering the fed-batch culture basal medium has an unconventionally high initial glucose concentration of 15 g/L, and an overall glucose consumption in excess of 17 g/L. The potential of this process platform was further demonstrated through additional media optimization, which has resulted in a final antibody concentration of 2.64 +/- 0.19 g/L and volumetric productivity of > 200 mg/L per day in a 13-day FBP for one of the five production cell lines. Use of this standardized protein-free, cholesterol-free NS0 FBP platform enables consistency in development time and cost effectiveness for manufacturing of therapeutic antibodies.     

Galectin‐3 deficiency protects pancreatic islet cells from cytokine‐triggered apoptosis in vitro

Beta cell apoptosis is a hallmark of diabetes. Since we have previously shown that galectin‐3 deficient (LGALS3^?/?) mice are relatively resistant to diabetes induction, the aim of this study was to examine whether beta cell apoptosis depends on the presence of galectin‐3 and to delineate the underlying mechanism. Deficiency of galectin‐3, either hereditary or induced through application of chemical inhibitors, β‐lactose or TD139, supported survival and function of islet beta cells compromised by TNF‐α + IFN‐γ + IL‐1β stimulus. Similarly, inhibition of galectin‐3 by β‐lactose or TD139 reduced cytokine‐triggered apoptosis of beta cells, leading to conclusion that endogenous galectin‐3 propagates beta apoptosis in the presence of an inflammatory milieu. Exploring apoptosis‐related molecules expression in primary islet cells before and after treatment with cytokines we found that galectin‐3 ablation affected the expression of major components of mitochondrial apoptotic pathway, such as BAX, caspase‐9, Apaf, SMAC, caspase‐3, and AIF. In contrast, anti‐apoptotic molecules Bcl‐2 and Bcl‐XL were up‐regulated in LGALS3^?/? islet cells when compared to wild‐type (WT) counterparts (C57BL/6), resulting in increased ratio of anti‐apoptotic versus pro‐apoptotic molecules. However, Fas‐triggered apoptotic pathway as well as extracellular signal‐regulated kinase 1/2 (ERK1/2) was not influenced by LGALS‐3 deletion. All together, these results point to an important role of endogenous galectin‐3 in beta cell apoptosis in the inflammatory milieu that occurs during diabetes pathogenesis and implicates impairment of mitochondrial apoptotic pathway as a key event in protection from beta cell apoptosis in the absence of galectin‐3. J. Cell. Physiol. 228: 1568–1576, 2013. © 2012 Wiley Periodicals, Inc.     

Anti-apoptotic genes Aven and E1B-19K enhance performance of BHK cells engineered to express recombinant factor VIII in batch and low perfusion cell culture

The engineering of production cell lines to express anti-apoptotic genes has been pursued in recent years due to potential process benefits, including enhanced cell survival, increased protein expression, and improved product quality. In this study, a baby hamster kidney cell line secreting recombinant factor VIII (BHK-FVIII) was engineered to express the anti-apoptotic genes Aven and E1B-19K. In high cell density shake flask culture evaluation, 11 clonal cell lines expressing either E1B-19K or a combination of Aven and E1B-19K showed improved survival compared to both parental and blank vector cell line controls. These cell lines exhibited lower caspase-3 activation and reduced Annexin-V binding compared to the controls. Parental and blank vector cell lines were less than 50% viable after 48 h of exposure to thapsigargin while cell lines expressing E1B-19K with or without Aven maintained viabilities approaching 90%. Subsequently, the best Aven-E1B-19K candidate cell line was compared to the parental cell line in 12-L perfusion bioreactor studies. Choosing the appropriate perfusion rates in bioreactors is a bioprocess optimization issue, so the bioreactors were operated at sequentially lower specific perfusion rates, while maintaining a cell density of 2 x 10(7) viable cells/mL. The viability of the parental cell line declined from nearly 100% at a perfusion rate of 0.5 nL/cell/day to below 80% viability, with caspase-3 activity exceeding 15%, at its lower perfusion limit of 0.15 nL/cell/day. In contrast, the Aven-E1B-19K cell line maintained an average viability of 94% and a maximum caspase-3 activity of 2.5% even when subjected to a lower perfusion minimum of 0.1 nL/cell/day. Factor VIII productivity, specific growth rate, and cell size decreased for both cell lines at lower perfusion rates, but the drop in all cases was larger for the parental cell line. Specific consumption of glucose and glutamine and production of lactate were consistently lower for the Aven-E1B-19K culture. Furthermore, the yield of ammonia from glutamine increased for the Aven-E1B-19K cell line relative to the parent to suggest altered metabolic pathways following anti-apoptosis engineering. These results demonstrate that expression of anti-apoptotic genes Aven and E1B-19K can increase the stability and robustness of an industrially relevant BHK-FVIII mammalian cell line over a wide range of perfusion rates.     

Akt‐mediated anti‐apoptotic effects of substance P in Anti‐Fas‐induced apoptosis of human tenocytes

Substance P (SP) and its receptor, the neurokinin‐1 receptor (NK‐1 R), are expressed by human tenocytes, and they are both up‐regulated in cases of tendinosis, a condition associated with excessive apoptosis. It is known that SP can phosphorylate/activate the protein kinase Akt, which has anti‐apoptotic effects. This mechanism has not been studied for tenocytes. The aims of this study were to investigate if Anti‐Fas treatment is a good apoptosis model for human tenocytes in vitro, if SP protects from Anti‐Fas‐induced apoptosis, and by which mechanisms SP mediates an anti‐apoptotic response. Anti‐Fas treatment resulted in a time‐ and dose‐dependent release of lactate dehydrogenase (LDH), i.e. induction of cell death, and SP dose‐dependently reduced the Anti‐Fas‐induced cell death through a NK‐1 R specific pathway. The same trend was seen for the TUNEL assay, i.e. SP reduced Anti‐Fas‐induced apoptosis via NK‐1 R. In addition, it was shown that SP reduces Anti‐Fas‐induced decrease in cell viability as shown with crystal violet assay. Protein analysis using Western blot confirmed that Anti‐Fas induces cleavage/activation of caspase‐3 and cleavage of PARP; both of which were inhibited by SP via NK‐1 R. Finally, SP treatment resulted in phosphorylation/activation of Akt as shown with Western blot, and it was confirmed that the anti‐apoptotic effect of SP was, at least partly, induced through the Akt‐dependent pathway. In conclusion, we show that SP reduces Anti‐Fas‐induced apoptosis in human tenocytes and that this anti‐apoptotic effect of SP is mediated through NK‐1 R and Akt‐specific pathways.     

Geniposide inhibits glucolipotoxicity and cooperates with Txnip knockdown to potentiate cell adaption to endoplasmic reticulum stress in pancreatic beta cells

Thioredoxin‐interacting protein (Txnip), a negative regulator of thioredoxin, has become an attractive therapeutic target to alleviate metabolic diseases. Our previous data demonstrated that geniposide improved glucose‐stimulated insulin secretion by accelerating Txnip degradation and prevented the early‐stage apoptosis of pancreatic β cells induced by palmitate, but the underlying mechanisms are still unclear. The objective of this study is to identify the role of Txnip in geniposide preventing the apoptosis of pancreatic β cells induced by high glucose and palmitate (HG/PA). The results revealed that geniposide attenuated HG/PA‐induced cell apoptosis and the expression of Bax and caspase‐3, while increasing mitochondrial membrane potential and the anti‐apoptotic protein levels of heme‐oxygenase‐1 (HO‐1) and Bcl‐2 in INS‐1 rat pancreatic β cells. Knockdown of the Txnip gene raised the levels of anti‐apoptotic proteins HO‐1 and Bcl‐2 and geniposide potentiated the effect of Txnip when the INS‐1 cells were challenged by HG/PA. Furthermore, geniposide enhanced the adoptive unfolded protein response by increasing the phosphorylation of PERK/eIF2α and IRE1α in HG/PA‐treated INS‐1 cells. The results together suggest that geniposide might be useful to antagonize glucolipotoxicity and Txnip might be a pleiotropic cellular factor in pancreatic β cells.     

Decreased expression of autophagic beclin 1 protein in idiopathic pulmonary fibrosis fibroblasts

Autophagy is the main cellular pathway for degradation of long‐lived proteins and organelles and regulates cell fate in response to stress. Beclin 1 is a key regulator of this process. In some settings autophagy and apoptosis seem to be interconnected. Recent reports indicate that fibroblasts in idiopathic pulmonary fibrosis (IPF) acquire resistance to apoptosis. Here, we examined the expression of beclin 1, and of the anti apoptotic protein Bcl‐2 in human IPF fibroblasts using immunohistochemistry and molecular biology in bioptic sections, in primary cultures of fibroblasts taken from patients with IPF and in fibroblast cell lines. Expression of beclin 1 in fibroblasts from IPF was down‐regulated in comparison with fibroblasts from normal lungs while the anti‐apoptotic protein Bcl‐2 expression was over‐expressed. Treatment of fibroblast cell cultures with cisplatin induced a significant increase in beclin 1 and caspase 3 protein levels but a reduction in Bcl‐2 expression. These observations were confirmed by the analysis of acid compartments and transmission electron microscopy. Our results demonstrate a modified expression of the apoptotic beclin 1 Bcl‐2 proteins in human IPF fibroblasts suggesting the existence of an autophagy/apoptosis system dysfunction. J. Cell. Physiol. 228: 1516–1524, 2013. © 2012 Wiley Periodicals, Inc.     

Betulinic acid induces apoptosis and suppresses metastasis in hepatocellular carcinoma cell lines in vitro and in vivo

Hepatocellular carcinoma (HCC) is a high incidence and mortality malignant tumour globally. Betulinic acid (BA) is a pentacyclic triterpenoid with potential pro‐apoptotic activities which widely found in many plants. In this study, we determined the effects of BA on proliferation, apoptosis, invasion, and metastasis in HCC cell lines and on tumour growth and pulmonary metastasis in mice. The results suggested that BA could inhibit cell viability and proliferation of HCC cell lines including HepG2, LM3, and MHCC97H. In addition, BA induced apoptosis of HepG2 cells characterised condensed nuclei and nuclear fragmentation. Moreover, western blot analysis showed that BA‐induced apoptosis associated with increasing of pro‐apoptotic protein Bax and cleaved caspase‐3 and decreasing of anti‐apoptotic protein Bcl‐2. Meanwhile, BA also reduced the reactive oxygen species (ROS) level. Furthermore, BA also significantly inhibited HCC growth in vivo and blocked pulmonary metastasis of HCC by regulating the metastasis‐related proteins including MMP‐2, MMP‐9, and TIMP2 without obvious toxicity. In all, the present study suggested that BA might be a promising anti‐HCC drug candidate by inhibiting proliferation, inducing apoptosis, and blocking metastasis.     

HIV‐1 promonocytic and lymphoid cell lines: an in vitro model of in vivo mitochondrial and apoptotic lesion

To characterize mitochondrial/apoptotic parameters in chronically human immunodeficiency virus (HIV‐1)‐infected promonocytic and lymphoid cells which could be further used as therapeutic targets to test pro‐mitochondrial or anti‐apoptotic strategies as in vitro cell platforms to deal with HIV‐infection. Mitochondrial/apoptotic parameters of U1 promonocytic and ACH2 lymphoid cell lines were compared to those of their uninfected U937 and CEM counterparts. Mitochondrial DNA (mtDNA) was quantified by rt‐PCR while mitochondrial complex IV (CIV) function was measured by spectrophotometry. Mitochondrial‐nuclear encoded subunits II–IV of cytochrome‐c‐oxidase (COXII‐COXIV), respectively, as well as mitochondrial apoptotic events [voltage‐dependent‐anion‐channel‐1(VDAC‐1)‐content and caspase‐9 levels] were quantified by western blot, with mitochondrial mass being assessed by spectrophotometry (citrate synthase) and flow cytometry (mitotracker green assay). Mitochondrial membrane potential (JC1‐assay) and advanced apoptotic/necrotic events (AnexinV/propidium iodide) were measured by flow cytometry. Significant mtDNA depletion spanning 57.67% (P < 0.01) was found in the U1 promonocytic cells further reflected by a significant 77.43% decrease of mitochondrial CIV activity (P < 0.01). These changes were not significant for the ACH2 lymphoid cell line. COXII and COXIV subunits as well as VDAC‐1 and caspase‐9 content were sharply decreased in both chronic HIV‐1‐infected promonocytic and lymphoid cell lines (<0.005 in most cases). In addition, U1 and ACH2 cells showed a trend (moderate in case of ACH2), albeit not significant, to lower levels of depolarized mitochondrial membranes. The present in vitro lymphoid and especially promonocytic HIV model show marked mitochondrial lesion but apoptotic resistance phenotype that has been only partially demonstrated in patients. This model may provide a platform for the characterization of HIV‐chronicity, to test novel therapeutic options or to study HIV reservoirs.