Knockdown of DEPTOR induces apoptosis, increases chemosensitivity to doxorubicin and suppresses autophagy in RPMI-8226 human multiple myeloma cells in vitro

Abstract

DEP domain containing mammalian target of rapamycin (mTOR)-interacting protein (DEPTOR) is an mTOR binding protein that is overexpressed in RPMI-8226 human multiple myeloma cells, and plays an important role in maintaining cell survival. However, knowledge on the effects of DEPTOR knockdown on the biological functions of RPMI‑8226 human multiple myeloma cells, is limited. This study aimed to determine the role of DEPTOR in the proliferation, apoptosis and autophagy in these cells and to elucidate the mechanisms by which DEPTOR contributes to the chemosensitivity of myeloma cells. RNA interference was used to reduce the expression of DEPTOR. Cytotoxicity was evaluated by MTT assay. Apoptosis was examined by flow cytometry. DEPTOR mRNA and protein expression in RPMI‑8226 cells treated with DEPTOR-specific short hairpin RNA (shRNA) was evaluated by RT-PCR, quantitative PCR and western blot analysis. The expression of apoptosis‑associated proteins, autophagy‑associated proteins, and the activation of the phosphoinositide 3‑kinase (PI3K)/Akt signaling pathway were detected by western blot analysis. Autophagy was also measured by transmission electron microscopy and monodansylcadaverine (MDC). In this study, RPMI-8226 cells were transfected with the DEPTOR-specific shRNA, which resulted in the significant inhibition of the transcription and expression of DEPTOR. The downregulation of DEPTOR inhibited proliferation, enhanced the doxorubicin‑induced growth inhibitory effects on RPMI-8226 cells, and increased the expression of cleaved caspase‑3 and cleaved poly(ADP-ribose) polymerase (PARP). Moreover, the downregulation of DEPTOR suppressed autophagy and inhibited the activation of the PI3K/Akt signaling in RPMI‑8226 cells. In conclusion, our data demonstrated that the downregulation of DEPTOR induces apoptosis, increases chemosensitivity to doxorubicin, and suppresses autophagy and the activation of the PI3K/Akt signaling pathway in RPMI‑8226 human multiple myeloma cells.

Lycorine: A prospective natural lead for anticancer drug discovery

“…Autophagy is a component of stress management system of the cells that is utilized by the cell for removal of defective organelles. Autophagy acts as a survival strategy in established and advanced tumors by maintaining cellular energy levels and enhances chemo-sensitivity of a number of anticancer agents to induce cell death [98][99][100][101][102]. It is not clear yet, how autophagy mediates the pro-survival effects in cancerous cells, the mechanism may involve limiting DNA-damage response-mediated apoptosis or, activation of the high-mobility group box 1 (HMGB1)/ receptor for advanced glycation end products (RAGE) signaling axis [103][104][105].…”

Deptor: not only a mTOR inhibitor

“…PI3K/Akt pathway is frequently hyper-activated in cancer and the activation of its downstream targets is associated with proliferation, differentiation and apoptosis [16]. Indeed, Akt mediates cell survival directly, by inhibiting pro-apoptotic proteins, such as caspase-9 and Bad, and indirectly, by modulating several regulators of cell death including p53 and Nf-KB [17]. In this scenario, high levels of Deptor can sustain cell survival in many cancer cell types.

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“…In addition, it has been recently demonstrated that Deptor stability is promoted by AMBRA1-mediated Cullin5 inhibition [17]. Moreover, through Deptor accumulation, AMBRA1 establishes a positive feedback loop reinforcing the inhibition of mTOR activity.…”

“…4) [2]. In particular, the absence of Deptor is sufficient to trigger caspase-dependent apoptosis [17]. Deptor suppression also induces a reduction in the PI3K/Akt activity, event that prevents the inhibitory phosphorylation of pro-caspase-3, thus promoting the apoptotic process (Fig.…”

“…Deptor suppression also induces a reduction in the PI3K/Akt activity, event that prevents the inhibitory phosphorylation of pro-caspase-3, thus promoting the apoptotic process (Fig. 4) [17]. …”