Won Jong Rhee

Silkworm hemolymph as a potent inhibitor of apoptosis in Sf9 cells

We have previously shown that silkworm hemolymph exhibits anti-apoptotic activity against baculovirus-induced Sf9 cell apoptosis. In this study, using various chemical inducers, such as actinomycin D, camptothecin, and staurosporine, we found that silkworm hemolymph inhibits insect cell apoptosis induced not only by baculovirus but also by chemical inducers. This indicates that silkworm hemolymph contains anti-apoptotic components that work directly in insect cell apoptosis without any booster expression of baculoviral genes. With the analysis of Sf-caspase-1 activity, it was found that the inhibitory effect of silkworm hemolymph works in a further upstream step than the Sf-caspase-1 activation step.

Inhibition of Human Cell Apoptosis by Silkworm Hemolymph

Many studies on preventing apoptosis have been carried out from the viewpoint of anti‐apoptotic cloned‐gene expressions inside cells, whereas in this study, we investigated the inhibition of apoptosis by the addition of silkworm hemolymph, a natural compound, from outside of the cells. In a previous study, we reported the inhibition effect of silkworm hemolymph on the baculovirus‐induced insect cell apoptosis. Using the vaccinia virus‐HeLa cell system as a model system in this study, we found that silkworm hemolymph, the insect serum, inhibits apoptosis not only in the insect cell system but also in the human cell system. The vaccinia virus‐induced HeLa cell apoptosis was analyzed using DNA electrophoresis, TUNEL, and flow cytometry, and the resulting data confirmed that silkworm hemolymph inhibits human cell apoptosis. The inhibition of apoptosis due to silkworm hemolymph was not caused by an inhibition of virus binding and internalization steps, nor did silkworm hemolymph interfere with the virus production. The inhibition of apoptosis by silkworm hemolymph decreased the cell detachment from an adhering surface. With these characteristics, silkworm hemolymph can be effectively used to minimize cell death in commercial animal cell culture.

Kinetic effect of silkworm hemolymph on the delayed host cell death in an insect cell-baculovirus system

The kinetic effect of silkworm hemolymph on host cell viability during a baculovirus‐induced insect cell death process was investigated. Host cell viability after viral infection is important for replication of the baculovirus DNA containing a recombinant gene and expression of the cloned gene. The baculovirus‐induced insect cell death process can be divided into a delay phase and a first‐order death phase, which are characterized by a delay time (td) and a specific death rate (kd), respectively. For 0–10% silkworm hemolymph in the media, higher concentrations resulted in longer delay times and lower specific death rates. By adding 10% silkworm hemolymph, the delay time increased from 72 to 164 h, and the specific death rate was reduced from 13.8 × 10−3 to 6.0 × 10−3 h−1. In addition, host cell viability correlated with DNA fragmentation, which is the biochemical hallmark of apoptosis. This indicates that the silkworm hemolymph inhibits the baculovirus‐induced insect cell apoptosis. However, the silkworm hemolymph did not affect the number of hypothetical targets, which represents host cell susceptibility to the baculovirus. The concentration of fetal bovine serum (FBS) in the medium did not affect the delay time, while lower concentrations of silkworm hemolymph resulted in shorter delay times. This means that the substance which increases the longevity of the host cell is not in the FBS but in the silkworm hemolymph.

Inhibition of Apoptosis by a Bombyx mori Gene

An apoptosis‐inhibiting component of silkworm hemolymph, isolated and characterized in our previous study, showed 95% N‐terminal amino acid sequence homology with one of the 30K proteins, a group of structurally related proteins. The 30K protein was expressed in mammalian HEK293 cells and CHOK1 cells by transfection with 30Kc6. The expression of 30Kc6inhibited apoptosis comparably to that of whole silkworm hemolymph, indicating that both intracellular expression and external supplementation inhibited apoptosis. The expression of 30Kc6resulted in lower intracellular activity for caspase 3. However, the results of in vitro assay of caspase 3 show that the 30Kc6 protein does not inhibit caspase 3 activity. This indicates that the 30Kc6 protein inhibits the apoptosis by working in a further upstream event than caspase 3 activation.

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 °C; however, the silkworm hemolymph heat‐treated at 70–80 °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 °C, while 30K protein lost its activity at temperatures higher than 60 °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.

Enhanced production of recombinant protein inEscherichia coli using silkworm hemolymph

The effect of silkworm hemolymph on the expression of recombinant protein inEscherichia coli was investigated. The addition of silkworm hemolymph to the culture medium increased the production of recombinant β-galactosidase inE. coli. The production was dependent on the concentration of the added silkworm hemolymph, which increased 2-, 5-, and 8-fold in media supplemented with 1,3, and 5% silkworm hemolymph, respectively. To identify the effective component, the silkworm hemolymph was fractionated by gel filtration column chromatography. A fraction, with a molecular weight of about 30 K was identified as the effective component.