Proteasome inhibitor MG-132 lowers gastric adenocarcinoma TMK1 cell proliferation via bone morphogenetic protein signaling

doi:10.1016/j.bbrc.2008.04.059

Biochemical and Biophysical Research Communications

Volume 371, Issue 2, 27 June 2008, Pages 209-214

https://doi.org/10.1016/j.bbrc.2008.04.059 Get rights and content

Abstract

Proteasome inhibitor is a novel class of cancer therapeutics, of which the mechanism of action is not fully understood. It is reported that proteasome inhibitor enhances bone morphogenetic protein (BMP) signaling in osteoblasts to stimulate bone formation. BMP signaling is also an important tumor-suppressing pathway in gastric carcinogenesis. We therefore sought to determine the anti-mitogenic effect of proteasome inhibition in relation to BMP signaling in gastric cancer cells. Results showed that proteasome inhibitor MG-132 significantly suppressed the proliferation and the colony-forming ability of gastric cancer TMK1 cells. In this connection, MG-132 activated BMP signaling, manifested as an increase in Smad1/5/8 phosphorylation and up-regulation of p21^Waf1/Cip1 mRNA and protein expression. Knockdown of BMP receptor II by RNA interference abolished Smad1/5/8 phosphorylation, p21^Waf1/Cip1 induction, and the inhibition of cell proliferation induced by MG-132. Further analysis revealed that MG-132 up-regulated the expression of BMP1 and BMP4 and suppressed the expression of Smad6. Knockdown of Smad6 also mimicked the effect of MG-132 on BMP signaling. Collectively, these findings suggest that inhibition of proteasome suppresses gastric cancer cell proliferation via activation of BMP signaling. This discovery may open up a novel therapeutic avenue to proteasome inhibitors for the management of gastric cancer.

Section snippets

Materials and methods

Reagents and drugs. The proteasome inhibitor MG-132 was purchased from Calbiochem (San Diego, CA). Antibodies for Smad1, phospho-Smad1/5/8, Smad6, and β-actin were purchased from Cell Signaling Technology (Beverley, MA, USA). Other primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals and reagents were purchased from Sigma–Aldrich (St. Louis, MO) unless otherwise specified.

Cell culture and viability assay. The human gastric adenocarcinoma

MG-132 inhibited TMK1 cancer cell proliferation and colony-forming ability

To study the effect of proteasome inhibitor on proliferation of gastric cancer cells, we examined changes in [³H]thymidine incorporation in response to MG-132 treatment in cultured gastric cancer TMK1 cells. In Fig. 1A, MG-132 significantly reduced TMK1 cell [³H]thymidine incorporation in a concentration- and time-dependent manner. At the dose of 1 μmol/L, 24-h treatment of MG-132 inhibited TMK1 cell proliferation by about 60%. The anti-mitogenic effect of MG-132 (1 mmol/L) could be detected as

Discussion

In mammals, the degradation of a repertoire of signaling molecules involved in cell cycle and apoptosis, such as p53, p27^Kip1, and I-κB, are regulated by the ubiquitin-proteasome pathway [2], [3], [4], [5], [6]. Inhibiting the proteasome results in the accumulation of these proteins and thereby promotes cell cycle arrest and apoptosis. It comes, therefore, as no surprise that the ubiquitin-proteasome pathway has become an attractive target of pathway-directed cancer therapy. A recent report

Acknowledgment

The study was supported by grants from the Hong Kong Research Grants Council (CUHK 7499/05M).

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Citation Excerpt :
These data suggest that activation of BMP signaling mediates part of the anti-cancer action of proteasome inhibitor. Further mechanistic investigation has revealed concomitant up-regulation of BMP ligands and down-regulation of Smad6 may be responsible for the activation of BMP signaling by proteasome inhibitor [11,100]. The mechanism by which proteasome inhibition regulates the expression of these genes at the transcriptional level remains unknown but it is speculated that proteasome inhibitors may inhibit the degradation of specific transcription factors that regulate the expression of BMP ligands and Smad6.
The ubiquitin–proteasome system is a major pathway for protein degradation. Targeting this pathway using proteasome inhibitors represents a novel approach for the treatment of cancer. Proteasome inhibitors lower cell proliferation and induce apoptosis in solid and hematologic malignancies through multiple mechanisms, including stabilization of cell cycle regulators and pro-apoptotic factors, stimulation of bone morphogenetic protein signaling, inhibition of protein translation, and sensitization to ligand-induced apoptosis. In this connection, proteasome inhibition activates macroautophagy, a compensatory protein degradation system, as well as other pro-survival signaling pathways. Inhibition of these auto-protective responses sensitizes cancer cells to the anti-proliferative effects of proteasome inhibitors.
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In this regard, proteasome inhibitor has been shown to induce the accumulation of p53, p27Kip1, p21Cip1, Bax, and I-κB, all of which are negative regulators of cell cycle or pro-apoptotic factors [19–23]. Moreover, proteasome inhibitor induces cell cycle arrest through the activation of bone morphogenetic protein signaling [24,25]. Proteasome inhibitor has been shown to down-regulate telomerase activity and survivin expression [26].
Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin–proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of ³⁵S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.
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The ubiquitin-proteosome system (UPS) has recently been the focus of several studies involved in the development of new cancer therapies [10–12]. The 26S proteosome is a large, multicatalytic threonine protease that provides the major pathway for degradation of ubiquitinylated intracellular proteins [10]. This 26S proteosome is involved in the targeted degradation of key regulatory proteins necessary for cell-cycle progression and apoptosis in normal and malignant cells.
Effective therapies for the subset of follicular thyroid cancer (FTC) patients with aggressive, metastatic disease are lacking. Therefore, we sought to determine the effects of proteosome inhibition, an emerging class of chemotherapeutic agents, on metastatic FTC cells.
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Macroautophagy plays a crucial part in the protein degradation system [15,16]. In this connection, inhibition of protein degradation, for example, by targeting the proteasome has been shown to lower cell proliferation or induce apoptosis in various types of epithelial and hematological malignancies [17–19]. Whether inhibition of macroautophagy can achieve similar effects on cancer growth, however, is unknown.
Macroautophagy is a process by which cytoplasmic content and organelles are sequestered by double-membrane bound vesicles and subsequently delivered to lysosomes for degradation. Macroautophagy serves as a major intracellular pathway for protein degradation and as a pro-survival mechanism in time of stress by generating nutrients. In the present study, bafilomycin A₁, a vacuolar type H⁺-ATPase inhibitor, suppresses macroautophagy by preventing acidification of lysosomes in colon cancer cells. Diminished macroautophagy was evidenced by the accumulation of undegraded LC3 protein. Suppression of macroautophagy by bafilomycin A₁ induced G₀/G₁ cell cycle arrest and apoptosis which were accompanied by the down-regulation of cyclin D₁ and cyclin E, the up-regulation of p21^Cip1 as well as cleavages of caspases-3, -7, -8, and -9 and PARP. Further investigation revealed that bafilomycin A₁ increased the phosphorylation of ERK, JNK, and p38. In this regard, p38 inhibitor partially reversed the anti-proliferative effect of bafilomycin A₁. To conclude, inhibition of macroautophagy by bafilomycin A₁ lowers G₁–S transition and induces apoptosis in colon cancer cells. Our results not only indicate that inhibitors of macroautophagy may be used therapeutically to inhibit cancer growth, but also delineate the relationship between macroautophagy and apoptosis.
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The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G₂/M cell cycle arrest which was associated with the formation of LC3⁺ autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3⁺ autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.
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Proteasome inhibitor MG-132 lowers gastric adenocarcinoma TMK1 cell proliferation via bone morphogenetic protein signaling

Abstract

Section snippets

Materials and methods

MG-132 inhibited TMK1 cancer cell proliferation and colony-forming ability

Discussion

Acknowledgment

Biochem. Biophys. Res. Commun.

Gastroenterology

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Cell

Cell

Protein degradation and protection against misfolded or damaged proteins

Nature

Bortezomib: proteasome inhibition as an effective anticancer therapy

Annu. Rev. Med.

In vivo ubiquitination and proteasome-mediated degradation of p53

Cancer Res.

Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27

Science

Identification of lysine residues required for signal-induced ubiquitination and degradation of I kappa B-alpha in vivo

Oncogene

Proteasome inhibition in the treatment of cancer

Cell Cycle

Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells

Nature