Protective Effect of the Silkworm Protein 30Kc6 on Human Vascular Endothelial Cells Damaged by Oxidized Low Density Lipoprotein (Ox-LDL)

Although the 30K family proteins are important anti-apoptotic molecules in silkworm hemolymph, the underlying mechanism remains to be investigated. This is especially the case in human vascular endothelial cells (HUVECs). In this study, a 30K protein, 30Kc6, was successfully expressed and purified using the Bac-to-Bac baculovirus expression system in silkworm cells. Furthermore, the 30Kc6 expressed in Escherichia coli was used to generate a polyclonal antibody. Western blot analysis revealed that the antibody could react specifically with the purified 30Kc6 expressed in silkworm cells. The In vitro cell apoptosis model of HUVEC that was induced by oxidized low density lipoprotein (Ox-LDL) and in vivo atherosclerosis rabbit model were constructed and were employed to analyze the protective effects of the silkworm protein 30Kc6 on these models. The results demonstrated that the silkworm protein 30Kc6 significantly enhanced the cell viability in HUVEC cells treated with Ox-LDL, decreased the degree of DNA fragmentation and markedly reduced the level of 8-isoprostane. This could be indicative of the silkworm protein 30Kc6 antagonizing the Ox-LDL-induced cell apoptosis by inhibiting the intracellular reactive oxygen species (ROS) generation. Furthermore, Ox-LDL activated the cell mitogen activated protein kinases (MAPK), especially JNK and p38. As demonstrated with Western analysis, 30Kc6 inhibited Ox-LDL-induced cell apoptosis in HUVEC cells by preventing the MAPK signaling pathways. In vivo data have demonstrated that oral feeding of the silkworm protein 30Kc6 dramatically improved the conditions of the atherosclerotic rabbits by decreasing serum levels of total triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC). Furthermore, 30Kc6 alleviated the extent of lesions in aorta and liver in the atherosclerotic rabbits. These data are not only helpful in understanding the anti-apoptotic mechanism of the 30K family proteins, but also provide important information on prevention and treatment of human cardiovascular diseases.     

Enhancement of recombinant protein production in Chinese hamster ovary cells through anti-apoptosis engineering using 30Kc6 gene

It was previously reported that silkworm hemolymph (SH) inhibits apoptosis and increases the production of recombinant human erythropoietin (EPO) in Chinese hamster ovary (CHO) cells. The apoptosis-inhibiting component in SH is a member of 30K protein family. In this study, the CHO cell line producing EPO was manipulated genetically to express the 30Kc6 gene encoding a 30K protein in the hemolymph of the silkworm, Bombyx mori. The transient expression of 30Kc6 significantly suppressed the cell death induced by serum deprivation. A stable cell line expressing 30Kc6 with an anti-apoptotic property was established. The stable expression of 30Kc6 inhibited serum-deprivation-induced apoptosis and increased the cell density and EPO titer by 5- and 10-fold, respectively. The positive effects of the 30Kc6 expression on cell viability and productivity were due to the stable maintenance of the mitochondrial activity. The 30Kc6 expression efficiently suppressed the depolarization of the mitochondrial membrane and subsequently balanced the generation/consumption of ATP. The use of the 30Kc6 gene is expected to provide a new method of host cell engineering for improving the productivity of the recombinant protein.     

Stabilization of enzymes by the recombinant 30Kc19 protein

In previous studies, we reported that the 30K protein originating from the silkworm inhibited apoptosis in mammalian cells. In this work, we demonstrated that the 30Kc19 protein, which is most abundant 30K protein in silkworm hemolymph, also enhanced enzyme stability. When the recombinant 30Kc19 protein was supplemented into distilled-deionized water containing alkaline phosphatase or horseradish peroxidase, deactivation of both enzymes induced by non-buffered DDW was significantly suppressed. The increase in enzyme stability due to the presence of 30Kc19 was similar to that observed for bovine serum albumin, which is commonly used in conventional enzyme reactions. The decrease in enzyme activity due to long-term storage in different buffer systems was also inhibited by 30Kc19. The 30Kc19 protein structure was shown to play a vital role in stabilizing the enzyme. These results imply that the recombinant 30Kc19 protein hold promise for use as an additive to increase or maintain enzyme activity.     

Expression of Bombyx mori 30Kc19 protein in Escherichia coli and its anti-apoptotic effect in Sf9 cell

The silkworm hemolymph has an anti-apoptotic activity in insect, mammalian, and human cell systems. The protein from silkworm hemolymph with the highest apoptosis inhibiting activity was found to be 30Kc19 protein, which was one of the ‘30K proteins’. In this study, 30Kc19 protein encoded by the 30Kc19 gene of the silkworm was expressed in Escherichia coli with (pET-22b(+)) and without (pET-3a) pelB leader sequence. 30Kc19 protein was over-expressed largely as a soluble form by pET-3a and both as soluble and insoluble forms by pET-22b(+). The medium was supplemented with each of the recombinant 30Kc19 proteins, and their presence was found to inhibit nuclear fragmentation and apoptotic body formation in actinomycin D-induced Sf9 cell apoptosis. Moreover, 30Kc19 protein repressed the activation of Sf-caspase-1. The 30Kc19 protein obtained from periplasm showed the most effective anti-apoptotic activity. This protein holds great potential for industrial and pharmaceutical applications since mass production and easy purification of this protein is possible.     

Enhancement of recombinant human EPO production and sialylation in chinese hamster ovary cells through Bombyx mori 30Kc19 gene expression

Productivity and sialylation are two important factors for the production of recombinant glycoproteins in mammalian cell culture. In our previous study, we found that silkworm hemolymph increased the sialylation of recombinant secreted human placental alkaline phosphatase in the insect cells, promoted the transfer of sialic acids onto the glycoprotein oligosaccharides in an in vitro asialofetuin sialylation system, and enhanced recombinant protein production in the Chinese hamster ovary (CHO) cells. These beneficial effects were mainly due to the 30K proteins, which consist of five isoforms. Among the 30K proteins, 30Kc19 was determined to be the major component. In this study, the 30Kc19 gene was introduced into a CHO cell line producing recombinant human erythropoietin, and its effects on productivity and sialylation were investigated. The transient expression of 30Kc19 significantly improved the production and sialylation of EPO. A stable cell line containing 30Kc19 was also established to investigate the effect of 30Kc19 gene expression. The stable expression of 30Kc19 increased the production and sialylation by 102.6% and 87.1%, respectively. The enhanced productivity from 30Kc19 expression is believed to occur because the 30Kc19 protein suppresses the loss of mitochondrial membrane potential and consequently improves the generation of intracellular ATP. In addition, the positive effect of 30Kc19 expression on sialylation is believed to be due to its ability to maintain sialyltransferase activity. In conclusion, 30Kc19 expression is a novel approach to improve the production and sialylation of recombinant glycoproteins in CHO cells.     

Enhancement of cell growth and viability of CHO cells in serum-free media by 30Kc6 gene expression

Many attempts have been made to develop a serum-free medium on account of the problems caused by serum in mammalian cell culture. However, serum deprivation inhibits cell growth and induces apoptosis. Moreover, adapting host cells to the serum-free medium is difficult and time-consuming. In a previous study, the anti-apoptotic 30K proteins were identified from silkworm hemolymph, which suggests that the 30K genes coding for the anti-apoptotic compound can be used for the anti-apoptosis engineering of mammalian cells. In this study, the 30K genes (30Kc6, 30Kc19, and 30Kc123) were introduced to DG44 CHO cells, which are the mammalian cell line most commonly used by industry for the production of biopharmaceuticals, in order to make them resistant to the apoptosis induced by serum deprivation. Among the 30K genes, the 30Kc6 gene exhibited the highest apoptosis-inhibition activity. When the 30Kc6-expressing cells cultivated in the serum-containing medium were transferred directly to commercially available serum-free media, 30Kc6 expression increased the viable cell density by four-fold through inhibiting serum deprivation-induced apoptosis.     

Inhibition of HeLa Cell apoptosis by storage-protein 2

Apoptosis is a barrier to maintaining high viable cell densities in animal cell culture. Silkworm hemolymph and its 30K protein have been reported to exhibit anti-apoptotic activity in various mammalian and insect cell systems. The 30K protein is thermally unstable at temperatures higher than 60 degrees C; however, the silkworm hemolymph heat-treated at 70-80 degrees C still exhibited anti-apoptotic activity. This indicates that silkworm hemolymph contains another anti-apoptotic compound other than 30K protein. In this article, the anti-apoptotic molecule other than 30K protein was found from the silkworm hemolymph and identified. This molecule was storage-protein 2 (SP2), which has no homology with any known anti-apoptotic protein. This molecule was heat-stable up to 80 degrees C, while 30K protein lost its activity at temperatures higher than 60 degrees C. When apoptosis was induced by staurosporine in HeLa cells, SP2 protein suppressed nuclear fragmentation and apoptotic body formation. Moreover, the generation of reactive oxygen species after apoptosis induction was inhibited, which means the inhibition occurred in an early step of the apoptotic process. Inhibition of apoptosis by the SP2 protein would lead to the minimization of cell death during commercial mammalian cell culture.     

Increase of 30K protein in identified motoneurons by hemolymph results in inhibition of programmed cell death in silkworm, Bombyx mori (Lepidoptera, Bombycidae)

This study demonstrates that a 30K protein was gradually synthesized in primary-cultured motoneurons from the accessory planta retractor (APR) of the 6th abdominal ganglion (APR6) in silkworm ventral ganglia through stimulation of hemolymph. An increase in 30K protein synthesis resulted in an inhibition of programmed cell death (PCD) of APR6 motoneurons. The 30K protein was gradually synthesized from the 30Kc6 gene of identified APR6s in day-6 4th instars to day-9 5th instar larvae, but synthesis of the 30K protein ceased in isolated APR6s of day-1 pupa, which normally begin to undergo PCD. When pupal APR6s were treated with larval hemolymph, however, the 30K protein was synthesized suggesting the existence of an anti-PCD factor in the larval hemolymph. An increase of 30K protein within the APR6s was confirmed by antiserum made against the recombinant 30K protein that originated from the APR 30Kc6 gene. Larval APR6, in which PCD was induced with 20-hydroxyecdysone (20E) added to the primary culture, exhibited some PCD characteristics of shrinkage of cell bodies, axonal fragmentation and loss of mitochondrial function. These results provide new insights on the survival or PCD of insect motoneurons through stimulation of hemolymph.     

Increase of host cell longevity by the expression of 30K protein originating from silkworm hemolymph in an insect cell–baculovirus system

A recombinant baculovirus was constructed by the homologous recombination between wild-type AcMNPV DNA and a baculovirus transfer vector containing a gene coding for the 30K protein originating from silkworm hemolymph. The 30K protein was successfully expressed in Sf9 cells infected with the recombinant baculovirus (AcMNPV/30K). To investigate the effect produced by the expression of the 30K protein, host cell viability after infection was compared with that of Sf9 cells infected with AcMNPV/β-gal. The viability of the cells infected with AcMNPV/β-gal began to decrease exponentially 3 days after infection, whereas that of the cells infected with AcMNPV/30K remained at a high level until 5 days after infection. This indicates that the 30K protein increases cell longevity after viral infection. This increased cell longevity is considered to be due to the inhibition of host cell apoptosis induced by a baculovirus, and the extent of apoptosis was measured by the flow cytometric method. The percentage of the sub-G1 fraction, which represents the extent of apoptosis, was decreased by the expression of the 30K protein. This indicates that the expression of the 30K protein in insect cells increases host cell longevity by inhibiting apoptosis.     

The homeodomain protein PBX participates in JH-related suppressive regulation on the expression of major plasma protein genes in the silkworm, Bombyx mori

In the silkworm, Bombyx mori, major plasma proteins referred to as 30K proteins are the most abundant proteins in the hemolymph of final (fifth) instar larvae. Surgical extirpation of corpora allata, the source of a juvenile hormone (JH), causes rapid accumulation of 30K proteins in the hemolymph of fourth instar larvae. The 30K protein 6G1 (30K6G1) gene was repressed in primary cultured fat body cells treated with a JH analog (JHA), methoprene. To identify the JH response element present in the promoter region of the 30K6G1 gene, we performed transfection analyses of the 5'-deletion mutants of the 30K6G1 gene using primary cultured fat body cells, gel retardation assays and in vivo footprinting analysis. The results from those analyses revealed that a JH response element exists in the sequence between positions -147 and -140. When the promoter construct mutated at positions -143, -142, and -141 was transfected to fat body primary cultured cells, the suppression effect on the reporter gene expression caused by JHA was reduced. Gel retardation assay using specific antibody revealed that a PBX protein binds to the JH response element. Northern blot analysis revealed that the gene expression of Bombyx PBX is enhanced in the fat body cells by JHA treatment. These results indicate that PBX proteins are involved in the JH signaling pathway and play an important role in suppressing 30K protein gene expression in the fat body of B. mori.     

Anti-oxidative effects of silkworm storage protein 1 in HeLa cell

Silkworm hemolymph contains unique proteins that exhibit anti-apoptotic activity in mammalian cells. Among them, 30 K protein, which is one of the major anti-apoptotic molecules in silkworm hemolymph, has been well investigated. However, little is known about the biological functions of storage protein 1 (SP1), another main protein in silkworm hemolymph. In this study, the anti-apoptotic and anti-oxidative activities of SP1 were analyzed. A stable cell line expressing SP1 was constructed, which showed strong anti-apoptotic effect induced by staurosporine treatment. In addition, the cell line exhibited resistance to oxidative stress caused by hydrogen peroxide. For practical applications of SP1, recombinant SP1 was produced in Escherichia coli, and the supplementation of recombinant SP1 into culture medium exhibited anti-apoptotic and anti-oxidative activities. In addition, SP1 was found to be a cell-penetrating protein and localized in the cytosol as well as on the plasma membrane. The findings showed that SP1 itself is not an anti-oxidant; rather, it mediates intracellular anti-oxidative activity. In conclusion, the cellular resistance of SP1 to apoptosis and oxidative stress will provide a new strategy that could be utilized in the bio-industry for the production of biologics as well as for the development of anti-aging cosmetics.     

Proteolytic specificity of caspases is required to signal the appearance of apoptotic morphology

The caspase family of cysteine proteases is essential for implementation of physiological cell death. Since a wide variety of cellular proteins is cleaved by caspases during apoptosis, it has been predicted that digestion of proteins crucial to maintaining the life of a cell is central to apoptosis. To assess the role of the proteolytic destruction during apoptosis, we introduced the non-specific protease proteinase K into intact cells. This introduction led to extensive digestion of cellular proteins, including physiological caspase-substrates. Caspase-3-like activity was induced rapidly, followed by morphological signs of apoptosis such as membrane blebbing and nuclear condensation. The caspase inhibitor Z-VAD-fmk inhibited the appearance of these morphological changes without reducing the extent of intracellular proteolysis by proteinase K. Loss of integrity of the cell membrane, however, was not blocked by Z-VAD-fmk. This system thus generated conditions of extensive destruction of caspase substrates by proteinase K in the absence of apoptotic morphology. Taken together, these experiments suggest that caspases implement cell death not by protein destruction but by proteolytic activation of specific downstream effector molecules.     

Antiapoptotic activity of 30 kDa lipoproteins family from fat body tissue of silkworm,Bombyx mori

The family of 30 kDa lipoproteins (LP1–5) is abundant in silkworm pupa fat body (FB) and hemolymph. One of its members, the 29 kDa protein decreased in concentration from peripheral (PP) FB tissue but was sustained in perivisceral (PV) FB tissue at the time of apoptosis. This study investigated the correlation of the 30 kDa proteins with FB apoptosis. Two protein fractions were purified, a 29 and a 30/31 kDa protein fraction, and they were used to test for activity against actinomycin D‐induced apoptosis in the FB tissues. Concentrations as little as 50 μg/mL of the 29 kDa protein fraction efficiently inhibited apoptosis. Less antiapoptotic activity was detected for the higher MW fraction; DNA fragmentation was observed in FB tissue treated with 50 μg/mL of the 30/31 kDa fraction. The viability of the cells in the 29 kDa protein‐supplemented culture was 40% higher than in the 31 kDa protein‐supplemented culture. However, the 30 kDa lipoproteins were not able to prevent scheduled FB degeneration during silkworm metamorphosis. Thus, it is hypothesized that the antiapoptotic 29 kDa protein needs to be proteolytically degraded by a regulatory mechanism to allow programmed cell death of FB tissue.     

Expression of anti-apoptotic 30Kc6 gene inhibiting hyperosmotic pressure-induced apoptosis in antibody-producing Chinese hamster ovary cells

In mammalian cell culture, elevating osmotic pressure can improve recombinant protein production by increasing the specific productivity. However, this operation also induces cell apoptosis. Thus, its beneficial effect is compromised. Previously, the expression of the 30Kc6 gene was found to inhibit apoptosis in Chinese hamster ovary (CHO) cells, resulting in an increase in recombinant protein production. In this study, the 30Kc6 gene was introduced into an antibody-producing CHO cell line, and its effect on hyperosmotic pressure-induced apoptosis was investigated. In the standard medium, the expression of 30Kc6 increased cell viability by 34.1% and productivity to 2.3 folds. After the osmotic pressure shift to 410 mOsm/kg, it was found that the viability of the 30Kc6-expressing cell decreased only by 8.5% as compared with that of the standard culture, while it decreased by 27.1% for the control cell. Consequently, the maximum production of the 30Kc6-expressing cell increased to 3.8 folds relative to that of the control cell in the standard condition. However the production rate did not increase for the control cell under the same conditions. 30Kc6 expression inhibited the hyperosmotic pressure-induced apoptosis at least partially because it repressed the mitochondrial membrane potential (MMP) reduction.     

Ramentaceone,a Naphthoquinone Derived from Drosera sp., Induces Apoptosis by Suppressing PI3K/Akt Signaling in Breast Cancer Cells

The phosphoinositide 3-kinase (PI3K) signaling pathway plays an important role in processes critical for breast cancer progression and its upregulation confers increased resistance of cancer cells to chemotherapy and radiation. The present study aimed at determining the activity of ramentaceone, a constituent of species in the plant genera Drosera, toward breast cancer cells and defining the involvement of PI3K/Akt inhibition in ramentaceone-mediated cell death induction. The results showed that ramentaceone exhibited high antiproliferative activity toward breast cancer cells, in particular HER2-overexpressing breast cancer cells. The mode of cell death induced by ramentaceone was through apoptosis as determined by cytometric analysis of caspase activity and Annexin V staining. Apoptosis induction was found to be mediated by inhibition of PI3K/Akt signaling and through targeting its downstream anti-apoptotic effectors. Ramentaceone inhibited PI3-kinase activity, reduced the expression of the PI3K protein and inhibited the phosphorylation of the Akt protein in breast cancer cells. The expression of the anti-apoptotic Bcl-2 protein was decreased and the levels of the pro-apoptotic proteins, Bax and Bak, were elevated. Moreover, inhibition of PI3K and silencing of Akt expression increased the sensitivity of cells to ramentaceone-induced apoptosis. In conclusion, our results indicate that ramentaceone induces apoptosis in breast cancer cells through PI3K/Akt signaling inhibition. These findings suggest further investigation of ramentaceone as a potential therapeutic agent in breast cancer therapy, in particular HER2-positive breast cancer.     

Insulin reduces apoptosis and increases DNA synthesis and cell size via distinct signalling pathways in Drosophila Kc cells

During development of Drosophila, cell proliferation and size are known to be regulated by insulin. Here we use Drosophila Kc cells to examine the molecular basis for the control of cell growth by insulin. Growing cells in the presence of insulin increased cell number above control levels at 16, 24, 48 and 72 h. We have demonstrated a novel anti-apoptotic effect of insulin (approximately 50%) in these cells, measured by caspase 3-like activity, which contributed to the increase in cell number. The anti-apoptotic effect was observed both in control cells and those in which apoptosis was induced by ultraviolet irradiation. An approximately 2-fold stimulation of bromodeoxyuridine incorporation demonstrated that insulin also increased Kc cell proliferation by stimulating new DNA synthesis. The ability of insulin to increase cell number, stimulate bromodeoxyuridine incorporation and reduce caspase 3-like activity was prevented by PD98059, which inhibits activation of the Drosophila extracellular signal regulated kinase (DERK) pathway, and was unaffected by wortmannin, an inhibitor of Drosophila phosphatidylinositol 3-kinase (DPI3K). Insulin also increased cell size approximately 2-fold and this was prevented by wortmannin and rapamycin, an inhibitor of Drosphilia target of rapamycin (DTOR). In summary, we show that DERK plays an important role in mediating the effect of insulin to reduce apoptosis and increase DNA synthesis whereas the DPI3K/DTOR/Dp70S6 kinase pathway mediates effects of insulin on cell size in Drosophila Kc cells.     

The stability and antiapoptotic activity of Bm30K-3 can be improved by lysine acetylation in the silkworm,Bombyx mori

Acetylation is an important, highly conserved, and reversible post-translational modification of proteins. Previously, we showed by nano-HPLC/MS/MS that many nutrient storage proteins in the silkworm are acetylated. Among these proteins, most of the known 30K proteins were shown to be acetylated, including 23 acetylated 30K proteins containing 49 acetylated sites (Kac), indicating the importance of the acetylation of 30K proteins in silkworm. In this study, Bm30K-3, a 30K protein containing three Kac sites, was further assessed in functional studies of its acetylation. Increasing the level of Bm30K-3 acetylation by adding the deacetylase inhibitor trichostatin A (TSA) increased the levels of this protein and further inhibited cellular apoptosis induced by H₂O₂. In contrast, decreasing the level of acetylation by adding the acetylase inhibitor C646 could reduce the level of Bm30K-3 and increase H₂O₂-induced apoptosis. Subsequently, BmN cells were treated with CHX and MG132, and increasing the acetylation level using TSA was shown to inhibit protein degradation and improve the stability of Bm30K-3. Furthermore, the acetylation of Bm30K-3 could compete with its ability to be ubiquitinated, suggesting that acetylation could inhibit the ubiquitin-mediated proteasome degradation pathway, improving the stability and accumulation of proteins in cells. These results further indicate that acetylation might regulate nutrition storage and utilization in Bombyx mori, which requires further study.     

Anti-apoptosis engineering

An increased understanding of apoptosis makes anti-apoptosis engineering possible, which is an approach used to inhibit apoptosis for the purpose of therapeutic, or industrial applications in the treatment of the diseases associated with increased apoptosis, or to improve the productivity of animal cell cultures, respectively. Some known anti-apoptotic proteins are the Bcl-2 family, IAP (inhibitor of apoptosis) and Hsps (heat shock proteins), with which anti-apoptosis engineering has progressed. This article reviews anti-apoptosis engineering using known anti-apoptotic compounds, and introduces a 30 K protein, isolated from silkworm hemolymph, as a novel anti-apoptotic protein, that shows no homology with other known anti-apoptotic proteins. The regulation of apoptosis, using anti-apoptotic proteins and genes originating from the silkworm,Bombyx mori, may provide a new strategy in this field.     

Molecular properties and tissue distribution of 30K proteins as ommin-binding proteins from diapause eggs of the silkworm, Bombyx mori

We previously reported the purification of an ommin-binding protein (OMBP) from an acid-methanol extract of diapause eggs of the silkworm and that OMBP reacted with the anti-30K proteins antiserum. In order to clarify the relationship between OMBP and the 30K proteins, we attempted to determine the sequence of the N-terminal amino acid of OMBP, which was separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). We observed ten protein spots of various isoelectric points; the spots corresponded with 30 kDa. Based on the sequence of the N-terminal amino acid (20 residues), the spots belonged to two kinds of 30K proteins (6G1 and 19G1), which are known as the major plasma proteins in the larval hemolymph of the silkworm. The proteins are expected to attach to polysaccharide because they reacted with concanavalin A and elderberry bark lectin. Immunohistochemical observations clarified that the proteins were localized in yolk granules and serosa in the diapause egg. These results suggest that OMBP is composed of 30K proteins which were modified with polysaccharides. In addition, the expression of 30K proteins mRNA was observed at early embryonic stage in diapause eggs by RT-PCR analysis. The 30K proteins as OMBP may play an important role in the transport and accumulation of tryptophan metabolites and ommochrome during the formation of serosa.