Staurosporine suppresses survival of HepG2 cancer cells through Omi/HtrA2-mediated inhibition of PI3K/Akt signaling pathway

Abstract

Staurosporine, which is an inhibitor of a broad spectrum of protein kinases, has shown cytotoxicity on several human cancer cells. However, the underlying mechanism is not well understood. In this study, we examined whether and how this compound has an inhibitory action on phosphatidylinositol 3-kinase (PI3K)/Akt pathway in vitro using HepG2 human hepatocellular carcinoma cell line. Cell viability and apoptosis were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxyribonucleotidyl transferase–mediated dUTP-digoxigenin nick end labeling (TUNEL) assay, respectively. Glutathione S-transferase (GST) pull-down assay and co-immunoprecipitation were performed to detect protein–protein interactions. Small interfering RNA (siRNA) was used to silence the expression of targeted protein. We found that staurosporine significantly decreased cell viability and increased cell apoptosis in a concentration- and time-dependent manner in HepG2 cancer cells, along with the decreased expressions of PDK1 protein and Akt phosphorylation. Staurosporine was also found to enhance Omi/HtrA2 release from mitochondria. Furthermore, Omi/HtrA2 directly bound to PDK1. Pharmacological and genetic inhibition of Omi/HtrA2 restored protein levels of PDK1 and protected HepG2 cancer cells from staurosporine-induced cell death. In addition, staurosporine was found to activate autophagy. However, inhibition of autophagy exacerbated cell death under concomitant treatment with staurosporine. Taken together, our results indicate that staurosporine induced cytotoxicity response by inhibiting PI3K/Akt signaling pathway through Omi/HtrA2-mediated PDK1 degradation, and the process provides a novel mechanism by which staurosporine produces its therapeutic effects.

Keywords

Staurosporine PDK1 apoptosis autophagy HepG2 cancer cells

Introduction

Unrestricted cell growth plays an important role in tumor development. Thus, modulation of cell death by targeting components of death machinery and its regulators is a rational approach for cancer therapy.^1–4 Usually, cell death is classified into at least four types: apoptosis, autophagic cell death, necrosis, and necrapoptosis.^5,6 Although the clear-cut distinctions among these types of cell death are ill-defined, classic apoptosis and autophagy still might be important targets for therapeutic intervention of cancer.

Staurosporine is a nonspecific protein kinase inhibitor isolated from Streptomyces staurosporeus exhibiting cytotoxicity activity.^7–10 Staurosporine can directly or indirectly trigger death pathway including apoptotic and necroptotic pathways,^9–13 leading to death of cancer cells. Moreover, staurosporine has been reported to regulate neuronal cell death through activation of autophagy.¹⁴ In HeLa cells, the impact of staurosporine on autophagy is dependent on its concentration. High-dose staurosporine (300 nmol/L) obviously impairs autophagic flux,¹⁵ suggesting that staurosporine is a regulator of autophagy in cancer cells. However, it has not been established whether autophagy is activated in hepatocellular carcinoma cells such as HepG2 cancer cells by staurosporine administration, and whether these processes affect survival of HepG2 cells.

Many signaling pathways have been reported to be involved in staurosporine-induced cell death or apoptosis of cancer cells, including cyclooxygenase-2 (COX-2) pathway,¹⁶ mitogen-activated protein kinase (MAPK) pathway,^17,18 Janus kinase (JAK)/(signal transducer and activator of transcription) STAT3 pathway,¹⁹ endoplasmic reticulum (ER) stress,²⁰ and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway.^21,22 Although a number of studies have evidenced that suppression of PI3K/Akt pathway may be functionally linked to the process of cell death induced by staurosporine,^23,24 the detailed mechanism underlying inhibition of PI3K/Akt signaling pathway remains largely unclear.

The serine protease Omi/HtrA2 is localized to mitochondria and translocates to the cytosol in response to apoptotic stimulation, where it increases degradation of X-linked inhibitor of apoptosis protein (XIAP). Overexpression of Omi/HtrA2 induces apoptosis in human cells, and its inhibition by RNA interference reduces cell death.^25,26 A number of studies have suggested the involvement of Omi/HtrA2 in development and progression of some types of cancers.^27–29 Several in vitro studies have also demonstrated that Omi/HtrA2 levels in the cytosol are associated with survival and differentiation of cells including cancer cells such as hepatocellular carcinoma cell lines, neuroblastoma cells, and human lung epithelial carcinoma cells.^30–32

In this study, we first demonstrate that staurosporine treatment induced Omi/HtrA2 release from mitochondria leading to PDK1 degradation and inhibition of downstream events such as phosphorylation of Akt and mTOR. Omi/HtrA2 was found to directly associate with PDK1. We further show that staurosporine-induced inhibition of mTOR activated autophagy. Moreover, pharmacological inhibition of autophagy exacerbated staurosporine-induced death of HepG2 cancer cells. Our data experimentally reveal that Omi/HtrA2 mediates cytotoxicity property of staurosporine.

Materials and methods

Antibodies and chemicals

Antibodies against HA-Tag (HA; H6908), glutathione S-transferase (GST; G7781), and α-tubulin (T9026) were from Sigma-Aldrich (St. Louis, MO, USA). Antibodies to Omi/HtrA2 (#9745), PDK1 (#13037), p-PDK1 Ser241 (#3061), Akt (#4691), p-Akt Ser473 (#9271), IRS-1 (#2382), p-IRS-1 Tyr895 (#3070), mTOR (#2983), and p-mTOR Ser2448 (#2971) were obtained from Cell Signaling Technology (Beverly, MA, USA). Protein A-sepharose beads were purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Staurosporine (S6942) was from Sigma-Aldrich. UCF-101 (#496150), bortezomib (BZM; #504314), and bafilomycin A1 (BFA, #196000) were purchased from Calbiochem (San Diego, CA, USA).

Cell culture

Human hepatocellular carcinoma HepG2 cells (ATCC, Rockville, MD, USA) were routinely cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in humidified atmosphere containing 5% CO₂ and 95% air at 37°C.

Plasmids construction

Vectors encoding HA-tagged wild-type full-length human Omi/HtrA2 (NM_001321727.1) or GST-Omi/HtrA2 fusion proteins in bacteria were constructed by subcloning polymerase chain reaction (PCR)-amplified DNA fragments into pcDNA3 (Invitrogen, Madison, WI, USA) or pGEX4T-1 (Amersham Pharmacia Biotech), respectively, as described previously.^33–35

Omi/HtrA2 small interfering RNA and transfection

Transfection was performed with 120 pmol/L small interfering RNA (siRNA) or its control using Lipofectamine^® RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.³⁶ The siRNA oligonucleotides against human Omi/HtrA2 were obtained from Qiagen (Shanghai, China) with following sequences: Omi/HtrA2 sense-1: 5′-CGGCUCAGGAUUCGUGGUGdTdT-3′, Omi/HtrA2 antisense-1: 5′-CACCACGAAUCCUGAGCCGdTdT-3′; Omi/HtrA2 sense-2: “5′-CACGAUCACAUCCGGCAUUdTdT-3′, Omi/HtrA2 antisense-2: 5′-AAUGCCGGAUGUGAUCGUGdTdT-3′; and scrambled control sense: 5′-AGCCAUCUGAUGCCGCAAAdTdT-3′, scrambled control antisense: 5′-UUUGCGGCAUCAGAUGGCUdTdT-3′.^33,37

Isolation of mitochondria

Mitochondria were prepared as previously described.^33,38 Briefly, cells were washed with ice-cold phosphate-buffered saline (PBS) and suspended in ice-cold isotonic buffer (containing 10 mmol/L HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 6.9, 200 mmol/L mannitol, 70 mmol/L sucrose, 1 mmol/L EDTA (ethylenediaminetetraacetic acid), and 1 mmol/L DTT (dithiothreitol)) supplemented with the protease inhibitor cocktail (P8340; Sigma-Aldrich). The cells were disrupted by brief sonication. Nuclei and unbroken cells were separated by centrifugation at 600g at 4°C for 10 min. The supernatant was further centrifuged at 14,000g at 2°C for 10 min to collect a mitochondrial pellet. The pellet was washed with the isotonic buffer and re-suspended into radioimmunoprecipitation assay buffer (RIPA) buffer (PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 1 mmol/L DTT) supplemented with the protease inhibitor cocktail described above.

Analysis of cell viability

Cell viability was confirmed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as our previously described protocol.³⁹ Briefly, 3 × 10³ cells/well were grown in DMEM with 20% FBS. After treatment with different concentration of staurosporine and/or indicated compounds for the desired time, the medium was replaced by 150 µL of fresh DMEM (without phenol red) with 50 µL of 0.5 mg/mL MTT solution. After incubation for 3 h at 37°C, the medium was carefully removed and 150 µL of MTT solvent (containing 4 mmol/L HCl, 0.1% Nonidet P-40, all in isopropanol) was added into each well. The plate was covered with tinfoil and agitated on an orbital shaker for 15 min. An ELISA (enzyme-linked immunosorbent assay) plate reader (BioTek, Winooski, VT, USA) was used to determine absorbance at 590 nm with a reference filter of 620 nm.

Apoptosis determination

Apoptosis was determined by terminal deoxyribonucleotidyl transferase–mediated dUTP-digoxigenin nick end labeling (TUNEL) assay. Briefly, the cells were fixed with 3.8% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 2 min, and then stained with the TUNEL reagent (Roche, Basel, Switzerland) for in situ apoptosis detection. Positive and negative controls were pretreated with 10 U/mL DNase or incubated without terminal deoxyribonucleotidyl transferase (TdT), respectively. Apoptotic cells were analyzed on an Olympus FluoView FV1000 confocal microscope (Olympus Imaging America Inc., Center Valley, PA, USA). The total number of intact cells and apoptotic cells (TUNEL positive) was counted in a random collection of 20% of the captured 40 time images. The apoptotic index was calculated by dividing the total number of apoptotic cells by the total number of intact cells and multiplying by 100.

GST pull-down, immunoprecipitation, and Western blot

Cell lysates were extracted by lysis buffer containing 50 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 20 mmol/L sodium pyrophosphate, 10 mmol/L NaF, 1 mmol/L NaVO₃, 2 mmol/L EDTA, 10% glycerol, 1 mmol/L MgCl₂, 1 mmol/L CaCl₂, 10% Nonidet-P40, 2 mmol/L phenylmethanesulfonyl fluoride (PMSF), and 10% phosphatase inhibitor cocktails 1, 2, and 3 (P2850, P5726, and P0044, respectively; Sigma-Aldrich). Protein concentration was determined using BCA (bicinchoninic acid) Protein Assay Reagent (Thermo, Rockford, IL, USA). For GST pull-down assay, 25 µg of GST or GST-Omi/HtrA2 fusion proteins immobilized on glutathione–agarose beads was incubated with 400 mL of cell lysates (containing ~800 µg of protein) overnight at 4°C.^33,36 For immunoprecipitation, cell lysates (containing ~500 µg of protein) were incubated overnight with specific antibodies bound to protein A-sepharose beads at 4°C.³⁶ After incubation, the beads were collected by centrifugation and washed extensively with ice-cold wash buffer containing 50 mmol/L HEPES, pH 7.6, 150 mmol/L NaCl, and 0.1% Triton X-100. The bound proteins were eluted by heating at 95°C for 10 min in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer. The proteins were separated by SDS-PAGE gel, transferred to a nitrocellulose membrane, and detected with specific antibodies.^36,39 Protein levels were normalized by α-tubulin levels or an internal reference protein from the same samples using Quantity One software (Bio-Rad, Philadelphia, PA, USA).

Statistical analysis

Data are presented as the mean ± standard deviation (SD). Differences between mean values were examined using one-way analysis of variance (ANOVA) followed by the Tukey–Kramer post hoc test and independent samples t test. The differences were considered statistically significant at p < 0.05. Each experiment in vitro was repeated at least three times with similar results. The quantification of the relative changes in protein expression and phosphorylation statuses was normalized with control protein expression in each experiment.

Results

Staurosporine-induced death of HepG2 cancer cells

To confirm the deleterious effects of staurosporine on survival of cancer cells, HepG2 cells were treated with 5, 10, 20, and 50 nmol/L staurosporine for 24 h, or 20 nmol/L staurosporine for 12, 24, and 48 h. Same volume of dimethyl sulfoxide (DMSO) served as the negative control. MTT assay and TUNEL assay were performed to detect cell viability and apoptosis, respectively. We found that staurosporine significantly reduced cell viability (Figure 1(a) and (b)) and markedly increased cell apoptosis (Figure 1(c) and (d); Supplementary Figure 1) in a concentration- and time-dependent manner.

Figure 1.

Staurosporine (STS)-induced cell death of HepG2 cancer cells: (a) concentration-response of STS on cell viability, (b) time-response of STS on cell viability, (c) concentration-response of STS on cell apoptosis, and (d) time-response of STS on cell apoptosis.

Staurosporine reduced intracellular PDK1 levels

Using the same protocol described as above, we next investigated the abundances and phosphorylation statues of some important signaling molecules in PI3K/Akt pathway in staurosporine-induced cells. Surprisingly, we found that staurosporine administration greatly inhibited PI3K/Akt pathway, as demonstrated by decreased protein levels and phosphorylation statues of PDK1 as well as decreased Akt phosphorylation (Figure 2(a) and (c)). Meanwhile, the levels of IRS-1 protein, IRS-1 phosphorylation, and Akt protein remained unchanged (Figure 2(a) and (c)). Staurosporine treatment reduced PDK1 levels in a concentration- and time-dependent manner (Figure 2(b) and (d)), suggesting that staurosporine induced PDK1 degradation.

Figure 2.

Staurosporine (STS) reduced intracellular PDK1 levels in HepG2 cancer cells: (a) concentration-response of STS on protein levels and phosphorylation of signaling molecules in PI3K/Akt pathway, (b) quantification of PDK1 levels in (a), (c) time-response of STS on protein levels and phosphorylation of signaling molecules in PI3K/Akt pathway, (d) quantification of PDK1 levels in (c), and (e) impacts of inhibitors of ubiquitin proteasome system and autophagy on PDK1 levels.

Ubiquitin proteasome system (UPS) and autophagy are two main routes of protein degradation.^40,41 To figure out the mechanism underlying PDK1 degradation, HepG2 cells were pretreated with 50 nmol/L of UPS-specific inhibitor BZM or 100 nmol/L of autophagy-specific inhibitor BFA for 1 h, and then incubated with 20 nmol/L staurosporine for 24 h. As shown in Figure 2(e), inhibition of both UPS and autophagy could not prevent staurosporine-induced PDK1 degradation. These data suggest that staurosporine suppressed PI3K/Akt signaling pathway by reducing PDK1 levels in an UPS- and autophagy-independent manner.

Omi/HtrA2 contributed to PDK1 degradation

As shown in Figure 3(a), Omi/HtrA2 levels in HepG2 cells treated with 20 nmol/L staurosporine for 24 h were significantly decreased in mitochondria, whereas the levels were markedly increased in supernatants. When HepG2 cells were pretreated with 20 µmol/L of Omi/HtrA2-specific inhibitor UCF-101 for 1 h and then incubated in 20 nmol/L staurosporine for another 24 h, we found that UCF-101 treatment significantly restored PDK1 levels, when compared with staurosporine treatment alone (Figure 3(b)). To further confirm these results, knockdown of Omi/HtrA2 in HepG2 cells was achieved using siRNA and then incubated in the presence or absence of 20 nmol/L staurosporine for 24 h. Consistently, Omi/HtrA2-knockdown cells showed higher PDK1 expression when compared with control cells under treatment with staurosporine (Figure 3(c) and (d)). These data suggest that staurosporine transported Omi/HtrA2 out of mitochondria leading to PDK1 degradation.

Figure 3.

Omi/HtrA2 was responsible for staurosporine (STS)-reduced PDK1 expression in HepG2 cancer cells: (a) Omi/HtrA2 release from mitochondria by STS, (b) effect of STS-specific inhibitor UCF-101 on PDK1 levels, and (c, d) impact of Omi/HtrA2 knockdown on PDK1 levels.

To understand the relationship between Omi/HtrA2 and PDK1, GST pull-down assay and co-immunoprecipitation assay were performed. As shown in Figure 4(a), Omi/HtrA2 fused with GST (GST-Omi), but not GST per se, interacted with PDK1. This interaction was enhanced by staurosporine stimulation. When HepG2 cells were treated with 20 nmol/L staurosporine for 12 h, we found that endogenous Omi/HtrA2 was co-immunoprecipitated with endogenous PDK1 (Figure 4(b)). The association between Omi/HtrA2 and PDK1 was decreased with staurosporine administration, due to Omi/HtrA2-reduced PDK1 levels. Indeed, overexpression of Omi/HtrA2 resulted in reduction of PDK1 levels (Figure 4(c)). These data indicate that Omi/HtrA2 physically interacted with PDK1 directly.

Figure 4.

Omi/HtrA2 associated with PDK1: (a) GST-Omi/HtrA2 bound with PDK1, (b) endogenous Omi/HtrA2 co-immunoprecipitated with endogenous PDK1 in HepG2 cancer cells, and (c) impact of overexpression of Omi/HtrA2 on PDK1 levels in HepG2 cancer cells.

Omi/HtrA2 inhibition attenuated the effects of staurosporine on cell death

To confirm important roles of Omi/HtrA2 in mediating staurosporine action, we next examined whether altering Omi/HtrA2 expression or activity affects staurosporine-stimulated death of HepG2 cancer cells. Wild-type or Omi/HtrA2-knockdown cells were pretreated with or without 20 µmol/L of Omi/HtrA2-specific inhibitor UCF-101 for 1 h and then incubated in 20 nmol/L staurosporine for another 24 h. MTT assay and TUNEL assay were performed to detect cell viability and apoptosis, respectively. We found that both pharmacological and genetic inhibition of Omi/HtrA2 significantly restored cell viability (Figure 5(a) and (b)) and suppressed cell apoptosis (Figure 5(c) and (d)). These results suggest that Omi/HtrA2 mediated staurosporine action on cell death.

Figure 5.

Omi/HtrA2 inhibition attenuated impacts of staurosporine (STS) on cell death of HepG2 cancer cells: (a) impact of Omi/HtrA2-specific inhibitor UCF-101 on cell viability, (b) impact of Omi/HtrA2 knockdown on cell viability, (c) impact of Omi/HtrA2-specific inhibitor UCF-101 on cell apoptosis, and (d) impact of Omi/HtrA2 knockdown on cell apoptosis.

Staurosporine activated autophagy

To investigate the potential effect of staurosporine on autophagy, HepG2 cells were treated with 20 nmol/L staurosporine for 6, 12, and 24 h. As shown in Figure 6, staurosporine significantly inhibited mTOR phosphorylation and increased expression of autophagy marker protein LC3-II (Figure 6(a) and (b)), suggesting that staurosporine effectively activated autophagy through suppression of mTOR. When HepG2 cells were pretreated with 20 µmol/L of Omi/HtrA2-specific inhibitor UCF-101 for 1 h and then incubated in the presence or absence of 20 nmol/L staurosporine for another 24 h, we found that UCF-101 treatment restored mTOR phosphorylation and suppressed staurosporine-stimulated LC3-II expression (Figure 6(c) and (d)). Consistently, Omi/HtrA2 knockdown exhibited similar results in HepG2 cells treated with 20 nmol/L staurosporine for 24 h when compared with scrambled control cells (Figure 6(e) and (f)). These data suggested that Omi/HtrA2 mediated activation of autophagy by staurosporine.

Figure 6.

Omi/HtrA2 modulated staurosporine (STS)-induced autophagy: (a) impact of STS on autophagy, (b) quantification of LC3-II levels in (a), (c) impact of Omi/HtrA2-specific inhibitor UCF-101 on autophagy, (d) quantification of LC3-II levels in (c), (e) impact of Omi/HtrA2 knockdown on autophagy, and (f) quantification of LC3-II levels in (e).

Autophagy inhibition exacerbated staurosporine-induced cell death

To confirm whether autophagy was involved in staurosporine-induced cell death and apoptosis, we used autophagy-specific inhibitor BFA to inhibit autophagy. HepG2 cells were pretreated with 100 nmol/L BFA for 1 h and then incubated in the presence or absence of 20 nmol/L staurosporine for another 24 h. BFA supplementation significantly enhanced the expression of LC3-II, suggesting autophagy inhibition (Figure 7(a)). Under this experimental condition, BFA administration further decreased staurosporine-induced cell viability and increased staurosporine-induced cell apoptosis (Figure 7(b) and (c)), suggesting that autophagy inhibition exacerbated staurosporine-induced cell death.

Figure 7.

Autophagy inhibition aggravated the impact of staurosporine (STS) on cell death of HepG2 cancer cells: (a) impact of autophagy inhibitor BFA on LC3-II expression, (b) impact of BFA on cell viability, and (c) impact of BFA on cell apoptosis.

Discussion

The PI3K/Akt signaling pathway plays a critical role in regulation of a number of cellular processes under physiological and pathophysiological conditions, including cell death and proliferation. Aberrant activation of this pathway in cancer cells is implicated with cellular transformation, tumorigenesis, cancer progression, and drug resistance.^42–44 Thus, inhibition or suppression of PI3K/Akt pathway has been identified as promising therapeutic targets for cancer therapy.^45–47

Previous studies have reported the potential impacts of staurosporine on PI3K/Akt pathway and demonstrated that this action is dependent on cell types. In small-cell lung carcinoma cells, staurosporine supplementation did not alter the constitutive activation pattern of the canonical PI3K/Akt signaling pathway.⁴⁸ However, reduction of phosphorylation status of Akt was found in U-937 leukemic cells,²³ neuroblastoma Neuro-2A cells,⁴⁹ and endometrial carcinoma (Ishikawa) cells,²⁴ in response to staurosporine stimulation. This reduction was functionally associated with staurosporine-induced cell apoptosis.^24,49 In agreement with these results, our results found that staurosporine administration effectively suppressed PI3K/Akt pathway, as evidenced by decreased phosphorylation of Akt (Figure 2) and mTOR (Figure 6), one of Akt downstream proteins. Importantly, we found that staurosporine treatment significantly decreased intracellular PDK1 levels in a concentration- and time-dependent manner (Figure 2). Meanwhile, its upstream protein IRS-1 remained at normal protein levels and phosphorylation statues (Figure 2), suggesting that PDK1 might be a targeted protein of staurosporine action. As an upstream kinase of Akt, PDK1 mediates PI3K/Akt pathway by phosphorylating Akt.^42,45 Undoubtedly, dysfunction or degradation of PDK1 would impede PI3K/Akt signaling pathway.^42,45,46

Usually, protein degradation is controlled mainly by two complicated interplay routes: UPS and autophagy.^40,41 A previous study has shown that PDK1 could be degraded in a proteasome-dependent manner.⁵⁰ However, our results found that specific inhibition of both UPS and autophagy did not alter staurosporine-reduced PDK1 expression (Figure 2(e)), suggesting that PDK1 degradation by staurosporine was caused by other mechanisms.

Omi/HtrA2 is a serine protease (also called a proteinase) mainly located in mitochondria, involving in both forms of apoptosis, caspase-dependent and caspase-independent cell death.^51,52 Upon apoptotic stimulation, Omi/HtrA2 translocates to the cytoplasm where it binds and cleaves inhibitor of apoptosis proteins and then initiates processes of cell death.^53–55 In addition, Omi/HtrA2 has also been found to facilitate the degradation of non-apoptotic proteins, leading to negative regulation of cell proliferation and activation of autophagy.^56,57 Consistent with this view, we found that staurosporine treatment significantly enhanced Omi/HtrA2 release from mitochondria (Figure 3(a)). Pharmacological and genetic inhibition of Omi/HtrA2 restored PDK1 levels reduced by staurosporine (Figure 3(b)–(d)). Overexpression of Omi/HtrA2 resulted in a decrease of PDK1 abundances (Figure 4(c)). Furthermore, GST pull-down assay and co-immunoprecipitation revealed that Omi/HtrA2 directly interacted with PDK1 (Figure 4(a) and (b)), which provided a structure mechanism for PDK1 cleavage. Hence, our findings strongly suggest that staurosporine-enhanced Omi/HtrA2 expression in cytosol was responsible for PDK1 degradation in HepG2 cancer cells. In addition, pharmacological and genetic inhibition of Omi/HtrA2 greatly mitigated the impacts of staurosporine on cell viability and apoptosis (Figure 5). This finding further confirms that Omi/HtrA2 mediated in vitro cytotoxicity of staurosporine.

Autophagy can be activated by staurosporine treatment or cytosolic accumulation of Omi/HtrA2.^14,57 However, the mechanism of how it mediates this is not well understood. It has been documented that mTORC1 is a major negative regulator of autophagy and the PI3K/Akt pathway is an upstream major modulator of mTORC1. Inhibition of PI3K/Akt/mTOR axis putatively stimulates autophagy.^58,59 Our results showed that Omi/HtrA2 accumulation in cytoplasm resulted in PDK1 degradation and subsequent inhibition of mTOR phosphorylation (Figures 2 and 6) and activation of autophagy (Figure 6). These findings provide a novel and rational explanation for both staurosporine- and Omi/HtrA2-stimulated activation of autophagy in HepG2 cancer cells.

A growing number of publications have shown that autophagy and apoptosis are interconnected by several molecular nodes of crosstalk.^60–62 The simultaneous activation of both phenomena has been found in vitro and in vivo under some physiological and pathophysiological conditions. Normally, both autophagy and apoptosis are recognized as tumor suppressor pathways.^61,62 However, autophagy appears to have dual impact for cancer development. Under certain circumstances, autophagy also facilitates the survival of tumor cells.⁶³ Thereby, autophagy is considered an important survival/protective mechanism for cancer cells.⁶² In this study, we found that autophagy inhibition exacerbated the impacts of staurosporine on cell viability and apoptosis (Figure 7),¹⁴ suggesting that autophagy plays a cell-protective role in staurosporine-treated HepG2 cells and that the autophagic process would be a promising target to regulate the development of cancer.

Taken together, our study has evidenced that staurosporine inhibited PI3K/Akt signaling pathway through Omi/HtrA2-mediated PDK1 degradation. We have also identified Omi/HtrA2 as a binding partner of PDK1 protein. This finding reveals a novel mechanism by which staurosporine suppressed the survival of cancer cells.

Footnotes

Acknowledgements

Youming Ding designed the research; Youming Ding, Bin Wang, Xiaoyan Chen, Yu Zhou, and Jianhui Ge performed the research; Bin Wang, Xiaoyan Chen, and Youming Ding analyzed the data; Xiaoyan Chen and Yu Zhou contributed new reagents and analytic tools; and Youming Ding wrote the paper.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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