Dataset Information

Extensive protein expression changes induced by pamidronate in RAW 264.7 cells as determined by IP-HPLC.

ABSTRACT:

Background

Bisphosphonate therapy has become a popular treatment for osteoporosis, Paget's disease, multiple myeloma, osteogenesis imperfecta, myocardial infarction, and cancer despite its serious side effects. Bisphosphonate-induced molecular signaling changes in cells are still not clearly elucidated.

Methods

As bisphosphonates are primarily engulfed by macrophages, we treated RAW 264.7 cells (a murine macrophage cell line) with pamidronate and investigated global protein expressional changes in cells by immunoprecipitation high performance liquid chromatography (IP-HPLC) using 218 antisera.

Results

Pamidronate upregulated proliferation-activating proteins associated with p53/Rb/E2F and Wnt/?-catenin pathways, but downregulated the downstream of RAS signaling, pAKT1/2/3, ERK-1, and p-ERK-1, and subsequently suppressed cMyc/MAX/MAD network. However, in situ proliferation index of pamidronate-treated RAW264.7 cells was slightly increased by 3.2% vs. non-treated controls. Pamidronate-treated cells showed increase in the expressions of histone- and DNA methylation-related proteins but decrease of protein translation-related proteins. NFkB signaling was also suppressed as indicated by the down-regulations of p38 and p-p38 and the up-regulation of mTOR, while the protein expressions related to cellular protection, HSP-70, NRF2, JNK-1, and LC3 were upregulated. Consequently, pamidronate downregulated the protein expressions related to immediate inflammation,cellular differentiation, survival, angiogenesis, and osteoclastogenesis, but upregulated PARP-1 and FAS-mediated apoptosis proteins. These observations suggest pamidronate affects global protein expressions in RAW 264.7 cells by stimulating cellular proliferation, protection, and apoptosis but suppressing immediate inflammation, differentiation, osteoclastogenesis, and angiogenesis. Accordingly, pamidronate appears to affect macrophages in several ways eliciting not only its therapeutic effects but also atypical epigenetic modification, protein translation, RAS and NFkB signalings. Therefore, our observations suggest pamidronate-induced protein expressions are dynamic, and the affected proteins should be monitored by IP-HPLC to achieve the therapeutic goals during treatment.

SUBMITTER: Lee SS

PROVIDER: S-EPMC7246033 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Extensive protein expression changes induced by pamidronate in RAW 264.7 cells as determined by IP-HPLC.

Lee Sang Shin SS Kim Soung Min SM Kim Yeon Sook YS Lee Suk Keun SK

PeerJ 20200521

<h4>Background</h4>Bisphosphonate therapy has become a popular treatment for osteoporosis, Paget's disease, multiple myeloma, osteogenesis imperfecta, myocardial infarction, and cancer despite its serious side effects. Bisphosphonate-induced molecular signaling changes in cells are still not clearly elucidated.<h4>Methods</h4>As bisphosphonates are primarily engulfed by macrophages, we treated RAW 264.7 cells (a murine macrophage cell line) with pamidronate and investigated global protein expres ...[more]

PMID: 32509464

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Project description:BackgroundCoffee extract has been investigated by many authors, and many minor components of coffee are known, such as polyphenols, diterpenes (kahweol and cafestol), melanoidins, and trigonelline, to have anti-inflammatory, anti-oxidant, anti-angiogenic, anticancer, chemoprotective, and hepatoprotective effects. Therefore, it is necessary to know its pharmacological effect on hepatocytes which show the most active cellular regeneration in body.MethodsIn order to determine whether coffee extract has a beneficial effect on the liver, 20 C57BL/6J mice were intraperitoneally injected once with dialyzed coffee extract (DCE)-2.5 (equivalent to 2.5 cups of coffee a day in man), DCE-5, or DCE-10, or normal saline (control), and then followed by histological observation and IP-HPLC (immunoprecipitation high performance liquid chromatography) over 24 h.ResultsMice treated with DCE-2.5 or DCE-5 showed markedly hypertrophic hepatocytes with eosinophilic cytoplasms, while those treated with DCE-10 showed slightly hypertrophic hepatocytes, which were well aligned in hepatic cords with increased sinusoidal spaces. DCE induced the upregulations of cellular proliferation, growth factor/RAS signaling, cellular protection, p53-mediated apoptosis, angiogenesis, and antioxidant and protection-related proteins, and the downregulations of NFkB signaling proteins, inflammatory proteins, and oncogenic proteins in mouse livers. These protein expression changes induced by DCE were usually limited to the range ± 10%, suggesting murine hepatocytes were safely reactive to DCE within the threshold of physiological homeostasis. DCE-2.5 and DCE-5 induced relatively mild dose-dependent changes in protein expressions for cellular regeneration and de novo angiogenesis as compared with non-treated controls, whereas DCE-10 induced fluctuations in protein expressions.ConclusionThese observations suggested that DCE-2.5 and DCE-5 were safer and more beneficial to murine hepatocytes than DCE-10. It was also found that murine hepatocytes treated with DCE showed mild p53-mediated apoptosis, followed by cellular proliferation and growth devoid of fibrosis signaling (as determined by IP-HPLC), and subsequently progressed to rapid cellular regeneration and wound healing in the absence of any inflammatory reaction based on histologic observations.

Project description:Although pentoxifylline (PTX) was identified as a competitive non-selective phosphodiesterase inhibitor, its pharmacological effect has not been clearly elucidated. The present study explored the effect of low dose 10 μg/mL PTX (therapeutic dose) compared to high dose 300 μg/mL PTX (experimental dose) in RAW 264.7 cells through immunoprecipitation-based high performance liquid chromatography (IP-HPLC), immunohistochemistry, and western blot. 10 μg/mL PTX increased the expression of proliferation (Ki-67, PCNA, cyclin D2, cdc25A), epigenetic modification (KDM4D, PCAF, HMGB1), protein translation (DOHH, DHPS, eIF5A1), RAS signaling (KRAS, pAKT1/2/3, PI3K), NFkB signaling (NFkB, GADD45, p38), protection (HSP70, SOD1, GSTO1/2), survival (pAKT1/2/3, SP1, sirtuin 6), neuromuscular differentiation (NSEγ, myosin-1a, desmin), osteoblastic differentiation (BMP2, RUNX2, osterix), acute inflammation (TNFα, IL-1, CXCR4), innate immunity (β-defensin 1, lactoferrin, TLR-3, -4), cell-mediated immunity (CD4, CD8, CD80), while decreased the expression of ER stress (eIF2α, eIF2AK3, ATF6α), fibrosis (FGF2, CTGF, collagen 3A1), and chronic inflammation (CD68, MMP-2, -3, COX2) versus the untreated controls. The activation of proliferation by 10 μg/mL PTX was also supported by the increase of cMyc-MAX heterodimer and β-catenin-TCF1 complex in double IP-HPLC. 10 μg/mL PTX enhanced FAS-mediated apoptosis but diminished p53-mediated apoptosis, and downregulated many angiogenesis proteins (angiogenin, VEGF-A, and FLT4), but upregulated HIF1α, VEGFR2, and CMG2 reactively. Whereas, 300 μg/mL PTX consistently decreased proliferation, epigenetic modification, RAS and NFkB signaling, neuromuscular and osteoblastic differentiation, but increased apoptosis, ER stress, and fibrosis compared to 10 μg/mL PTX. These data suggest PTX has different biological effect on RWA 264.7 cells depending on the concentration of 10 μg/mL and 300 μg/mL PTX. The low dose 10 μg/mL PTX enhanced RAS/NFkB signaling, proliferation, differentiation, and inflammation, particularly, it stimulated neuromuscular and osteoblastic differentiation, innate immunity, and cell-mediated immunity, but attenuated ER stress, fibrosis, angiogenesis, and chronic inflammation, while the high dose 300 μg/mL PTX was found to alleviate the 10 μg/mL PTX-induced biological effects, resulted in the suppression of RAS/NFkB signaling, proliferation, neuromuscular and osteoblastic differentiation, and inflammation.

Dataset Information

Extensive protein expression changes induced by pamidronate in RAW 264.7 cells as determined by IP-HPLC.

Background

Methods

Results

Publications

Extensive protein expression changes induced by pamidronate in RAW 264.7 cells as determined by IP-HPLC.

Similar Datasets

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