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Cell Biology International (2012) 36, 893–899 (Printed in Great Britain)
The apoptotic effect and associated signalling of HSP90 inhibitor 17-DMAG in hepatocellular carcinoma cells
Ai‑min Leng*1, Ting Liu*1, Jing Yang*, Jian‑fang Cui*, Xiu‑hua Li*, Ya‑nan Zhu*, Ting Xiong*, Guiying Zhang*2 and Yuxiang Chen†2
*Department of Gastroenterology, Xiangya Hospital, Central South University, Changsha 410008, People's Republic of China, and †School of Biological and Technology, Central South University, Changsha 410008, People's Republic of China


Primary liver cancer is one of the highly malignant tumours. The traditional surgery, chemotherapy and radiation therapy only established 6% of 5-year survival rate in HCC (hepatocellular carcinoma). Therefore there is an urgent need to develop new therapeutic strategies. HSP90 (heat shock protein 90) is one of the important molecular chaperones and was identified with high expression in the primary liver cancer. In this study, we evaluated the therapeutic effect of specific HSP90 inhibitor 17-DMAG (17-dimethylaminoethylamino-17-demethoxy geldanamycin) in HCC cells. The time and concentration effects of 17-DMAG were investigated in HCC cells. Cell proliferation was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and cell counting. Apoptosis was detected by flow cytometry with staining of Annexin V-FITC/PI (propidium iodide). The protein levels of survivin, cyclin D1, p53 and NF-κB (nuclear factor κB) were measured by Western blotting. 17-DMAG inhibited the proliferation of HCC cells in a time- and concentration-dependent manner. Treatment with 400 nmol/l 17-DMAG for 48 h significantly induced early-stage apoptosis (22.4%). Conversely, it induced less late-stage apoptosis (3.03%). The 5 mg/l of cisplatin induced significantly less early-stage apoptosis (6.5%), but similar proportion of late-stage apoptosis (4.89%) compared with 17-DMAG. Inhibition of HSP90 activity by 400 nmol/l 17-DMAG decreased protein levels of survivin, cyclin D1 and NF-κB protein levels, whereas increased p53 protein level. HSP90 plays a key role in HCC cell growth and survival through regulation of survivin, cyclin D1, p53 and nucleus NF-κB protein levels and the specific HSP90 inhibitor 17-DMAG can play a therapeutic role in HCC treatment.


Key words: cisplatin, heat shock protein 90, hepatocellular carcinoma, 17-DMAG

Abbreviations: 17-AAG, 17-allylamino derivative of geldanamycin, 17-DMAG, 17-dimethylaminoethylamino-17-demethoxy geldanamycin, CTD, C-terminal domain, DTT, dithiothreitol, HCC, hepatocellular carcinoma, HSP90, heat shock protein 90, IKK, IκB kinase, MDM2, murine double minute-2, MDMX, murine double minute-X, MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, NF-κB, nuclear factor κB, PI, propidium iodide, PS, phosphatidylserine

1These authors contributed equally to the study.

2Correspondence may be addressed to either of these authors (email chenyx008@yahoo.com or zhangguiying2007@gmail.com).


1. Introduction

HCC (hepatocellular carcinoma) is a primary liver cancer and is the fifth most common cancer worldwide with over 620000 newly diagnosed cases annually. Its incidence is rapidly increasing worldwide and HCC-related deaths ranks third among all cancers (Maillard, 2011). China accounts for 11000 patient deaths annually (Xu et al., 2010). Surgical resection is the most effective way to treat primary liver cancer. However, most patients present at an advanced, inoperable stage. These patients have an extremely poor prognosis and require other types of treatment (Ades, 2009). Palliative treatments, such as hepatic arterial chemoembolization, percutaneous ethanol injection, systemic chemotherapy as well as radiation produced a noticeable improvement in the patient's health, but the overall 5-year survival rate of HCC is only approximately 6% (Frangov et al., 2007). Therefore developing more effective and specific therapeutic strategies should be of the highest priority in HCC research. With the identification of new biological markers of cancer, molecular-targeted therapy has become a critical strategy for the HCC treatment.

HSP90 (heat shock protein 90) consists of two monomers with three domains each: a conserved N-terminal domain, CTD (C-terminal domain) and middle domain. The CTD contains binding sites for many substrates, including the CaM (calmodulin) binding site. The N-terminal domain is the ATP/ADP-binding site, rendering HSP90 ATP hydrolysis activity (Obermann et al., 1998). HSP90 protein forms a multi-chaperone complex with its clients to help correct folding, stabilize conformation and prevent degradation of substrate proteins (Hartl, 1996). Recent studies have found that HSP90 plays an important role in tumorigenesis and tumour progression. Particularly, several key HSP90 clients are involved in invasion, angiogenesis and metastasis of tumours (Stingl et al., 2010). The diverse molecular functions of HSP90 suggest that its inhibitors could possibly provide a promising strategy for tumour-target therapy. High expression of HSP90 was observed in primary liver cancer and adjacent normal liver tissues (Tommasi et al., 2007). Moreover, HSP90 is closely related to phosphorylation and proteasomal degradation of key proteins in primary liver cancer (Lee et al., 2003). Therefore inhibition of HSP90 activity could be an ideal strategy to inhibit HCC progression.

Several chemically distinctive and highly specific HSP90 inhibitors have been tested and showed compelling anticancer activity (Workman et al., 2007). The 17-DMAG (17-dimethylaminoethylamino-17-demethoxy geldanamycin) is a semi-synthetic derivative of geldanamycin, which can specifically target the ATP/ADP-binding sites at the C-terminus of HSP90, and competitively inhibits the ATP activity of HSP90 and abolish its chaperone function. Moreover, 17-DMAG can destabilize substrate proteins and block tumour survival signalling networks. Therefore 17-DMAG can inhibit cell proliferation and induce apoptosis, which results in the inhibition of tumour growth. Interestingly, a clinical trial showed that 17-DMAG is widely distributed in human tissues, while the residue 17-DMAG was only detected in tumour tissue 48 h after the administration, suggesting that it can specifically bind to tumour cells (Ayrault et al., 2009). Recently, the anti-tumour activity of 17-DMAG has been observed in medulloblastoma (Babchia et al., 2008), melanoma (Lang et al., 2007) and pancreatic tumour cells (Tommasi et al., 2007). The therapeutic effect of 17-DMAG is achieved by inducing cell apoptosis, inhibiting tumour invasion and increasing sensitivity of tumour cells to chemotherapeutic drugs. However, the role of 17-DMAG in the HCC cells has yet to be reported, particularly, its anti-tumour mechanisms have not yet been fully elucidated.

In this study, we investigated the time-dependent and concentration-dependent nature of 17-DMAG in inhibiting HCC cell proliferation and inducing apoptosis. We further investigated the effect of 17-DMAG on some critical molecules of the HSP90 signalling network. A significant therapeutic effect of 17-DMAG was seen in HCC cells.

2. Materials and methods

2.1. Cell culture

HepG2, a human liver HCC cell line, was obtained from ATCC and cultured in DMEM (Dulbecco's modified Eagle medium; Invitrogen) with 10% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C, 5% CO2.

2.2 MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] colorimetric assay

Cell viability was analysed by Thiazolyl Blue (MTT; Sigma–Aldrich). The MTT assay examines the activity of metabolic enzymes in the mitochondria of live cells. Therefore MTT can reflect cell proliferation. Cells that were grown to 50–60% confluency in 96-well plates were treated with 17-DMAG at a final concentration of 0, 100, 250, 500 and 1000 nmol/l. Cells treated with 5 mg/l cisplatin (DDP) were used as positive control. After incubation for 24, 48 and 72 h, cells were subjected to an absorbance reading at 490 nm using a 96-well microplate reader. The absorbance values were normalized to cells treated with 0 nmol/l 17-DMAG.

2.3. Cell morphology and number counting

HepG2 cells were seeded in 96-well plates at a density of 1×104 cells/well. After incubation for 24 h, cells were treated with 17-DMAG at a final concentration of 0, 100, 250, 500 and 1000 nmol/l. Cells treated with 5 mg/l cisplatin (DDP) were used as positive control. After continuous incubation for 24, 48 and 72 h, cells were observed under inverted microscope. To count the increased cell number, HepG2 cells were then trypsinized and cell number was counted using haemocytometer by two researchers blinded to the experimental design. The increased cell number was calculated as percentage of the cells treated with 0 nmol/l 17-DMAG.

2.4. Flow cytometry assay of cell apoptosis

PS (phosphatidylserine) is located in the inner membrane of a cell's lipid bilayer and it is transferred to the outside of the membrane after apoptosis occurs. Annexin V is a calcium-dependent phospholipid-binding protein, which can specifically bind to PS exposed to the outer side of the membrane. Using fluorescein (FITC) conjugated Annexin V (Keygen Company), the stained apoptotic cells can be counted. PI (propidium iodide) is a dye for nucleic acid but can only penetrate into the later stages of apoptotic cells. Therefore using both Annexin V and PI, apoptosis at different stages can be distinguished. For example, Annexin+/PI means the early apoptotic cells, Annexin+/PI+ means the late apoptotic cells and Annexin/PI means normal cells.

HepG2 cells were seeded in 6-well plates at 2×105 cells/well. After cells were grown to 70–80% confluency, they were treated with 0 and 400 nmol/l 17-DMAG and continuously incubated for 48 h. Cells treated with 5 mg/l DDP were used as positive control. The cells were then trypsinized and suspended into solution containing Annexin V-FITC and PI. After incubation for 1 h in the dark, cells were subjected to flow cytometry assay.

2.5. Nuclear fractionation

HepG2 cells were seeded into 10 cm dishes at 1×106 cells/well and treated with 0 and 400 nmol/l 17-DMAG for 6, 12 or 24 h. Nuclear fractionation was performed as previously described (Mi et al., 2006). Briefly, nuclear extracts were obtained by incubating cells with hypotonic buffer A [20 mM Hepes, pH 7.0, 10 mM KCl, 1 mM MgCl2, 10% glycerol, 0.5 mM DTT (dithiothreitol) and 0.25 mM PMSF] for 10 min on ice. The lysates were centrifuged at 200 g for 10 min and the pellet was then washed thrice with buffer A. To extract nuclear proteins, 100 μl of buffer A containing 300 mM NaCl and 0.1% NP-40 was finally applied to the pellet. The NF-κB (nuclear factor κB) protein level in the nucleus was monitored by anti-human NF-κB p50 antibody (Boster Inc.).

2.6. Cell homogenate and Western blotting

HepG2 cells were seeded in 10 cm dishes at 1×106 cells/well. After cells were grown to 70–80% confluency, they were treated with 0 or 400 nmol/l 17-DMAG and continuously incubated for 6, 12 or 24 h. Cells were then harvested and homogenized in cell lysis buffer containing: 50 mM Tris/HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 100 μg/ml PMSF and 1 mM DTT. Western blotting was performed as previously described (Zhang et al., 2003). The antibodies for human survivin, p53, β-actin, cyclin D1 and HRP (horseradish peroxidase)-conjugated second antibodies were purchased from Boster Inc. To control for loading efficiency, the blots were stripped and reprobed with β-actin. The images were scanned with Adobe Photoshop and quantified with NIH Image J. Expression levels of p53, cyclin D1 and survivin proteins were evaluated relative to that of β-actin protein where described (i.e. relative density = protein/β-actin levels). Background correction values were subtracted from each lane to minimize the variability across membranes.

2.7. Statistical analysis

Data were analysed using Statistical Package for the Social Science, version 16.0, and one-way ANOVA followed by Bonferroni paired t test were used to assess statistical significance of difference between treatment groups. A P<0.05 was considered statistically significant difference.

3. Results

3.1. 17-DMAG inhibited HepG2 cell proliferation in time- and concentration-dependent manner

HepG2 cells were treated with 0, 100, 250, 500 or 1000 nmol/l of 17-DMAG for 24, 48 or 72 h, respectively. Cell proliferation was measured by MTT assay. Results showed that 17-DMAG significantly inhibited cell proliferation in a time- and concentration-dependent manner (Figure 1). 17-DMAG (100 nmol/l) exhibited similar efficacy as 5 mg/l cisplatin (DDP) did. The IC50 for 24, 48 and 72 h is 1057, 406 and 302 nM, respectively. Under inverted microscope, cells treated with different concentrations of 17-DMAG for 24, 48 and 72 h showed unhealthy, such as uneven cell density, small and round shape and detachment. The changes are also time and concentration dependent (Figure 2). The cell number counting of small and round cells showed that 17-DMAG inhibited HepG2 cell proliferation in a time- and concentration-dependent manner.

3.2. 17-DMAG-induced apoptosis in HepG2 cells

The changes in morphology suggested that inhibition of HSP90 activity might induce cell death. We further performed Annexin-FITC/PI double staining. As shown in Figure 3, 400 nmol/l 17-DMAG induced significantly more early-stage apoptosis in HepG2 cells (22.42±1.83%), which is more effective than 5 mg/l cisplatin (6.5±1.20%). In contrast, 17-DMAG induced less late-stage apoptosis (3.03±0.70%), which is similar to the effect of 5 mg/l cisplatin (4.89±0.69%).

3.3. 17-DMAG inhibited survivin, cyclin D1 and NF-κB, but decreased p53 protein levels in HepG2 cell

We further measured the levels of survivin, cyclin D1, NF-κB and p53 proteins using Western blots. Treatment with 400 nmol/l 17-DMAG for 6, 12 or 24 h significantly decreased NF-κB, survivin and cyclin D1 protein levels, but increased p53 protein level (Figure 4A). These results suggested that inhibition of HSP90 activity down-regulated NF-κB, survivin and cyclin D1 protein levels while up-regulating total p53 protein level.

4. Discussion

Primary liver cancer deals with highly malignant tumours of liver cells or bile-duct cells. Traditional surgery, chemotherapy or radiotherapy offer limited therapeutic effects. High recurrence rates were reported even after radical resection. With extensive progress in molecular biology, genomics and proteomics, molecular target therapy of liver cancer has become possible. High expression of HSP90 in tumour cells and its molecular chaperone function suggested that HSP90 is a promising target for tumour therapy. In this study, we found that the specific HSP90 inhibitor 17-DMAG can time and concentration dependently inhibit HCC cell proliferation and induce cell apoptosis. The inhibition of HSP90 activity and subsequent induction of apoptosis are associated with down-regulation of survivin, cyclin D1 and NF-κB levels as well as up-regulation of p53 protein level. Our study indicates that targeting HSP90 is a promising strategy for the therapy of HCC.

The 17-AAG (17-allylamino derivative of geldanamycin) is the main HSP90-specific inhibitor tested in clinical trials and has demonstrated anti-tumour activity in a variety of tumours including prostate, breast and multiple myeloma (http://www.nci.nih.gov/clinicaltrials). However, 17-AAG is hindered by a number of limitations in clinical trials, such as its complex and cumbersome formulation, poor solubility and lack of oral bioavailability (Sharp and Workman, 2006). With more progress in developing additional geldanamycin analogues, the more soluble 17-DMAG was found with many advantages over 17-AAG (Egorin et al., 2002). The 17-DMAG has similar activity to 17-AAG both in vitro and in vivo, but is more water soluble and orally bioavailable (Kaur et al., 2004; Smith et al., 2005). Importantly, 17-DMAG at a dose of 75 mg/kg has been shown to distribute itself widely in tissues, but was retained longer in tumours than normal tissues in SCID (severe combined immunodeficiency) mice-bearing MDA-MB-231 human breast cancer xenografts (Eiseman et al., 2005). In this study, 17-DMAG inhibited HCC cell proliferation and induced apoptosis in a time- and concentration-dependent manner. A 100 nmol/l 17-DMAG was more effective than the 5 mg/l cisplatin, a widely used anticancer drug. Cisplatin has also been reported to bind to the putative ATP/novobiocin-binding site, near to the C-terminal of HSP90 and subsequently affect its chaperone function (Itoh et al., 1999; Rosenhagen et al., 2003). Our study suggested that 17-DMAG should be a great candidate for HCC treatment.

HSP90 was demonstrated to physically interact with and stabilize survivin in cells by binding to the mature form of survivin (Fortugno et al., 2003a, 2003b; Gyurkocza et al., 2006; Siegelin et al., 2009). With the importance of survivin in cancer survival and progression, various research groups have attempted to target survivin using HSP90 inhibitors. HSP90 inhibitors such as geldanamycin, 17-AAG and shepherdin have been shown effective in targeting the HSP90/survivin complex and subsequently inducing proteasomal degradation of survivin (Fortugno et al., 2003a 2003b; Gyurkocza et al., 2006; Siegelin et al., 2009). However, one study revealed that 17-AAG induced the overexpression of survivin in three different human cancer cell lines (Cheung et al., 2010). This suggested that regulation of HSP90 on survivin expression is tumour cell-type specific. Our study first demonstrated that 17-DMAG significantly down-regulated survivin protein level in HCC cells. Cyclin D1 is another key factor involved in cell proliferation. Inhibition of HSP90 activity with 17-AAG has been shown to down-regulate cyclin D1 expression in Mantle cell lymphoma and Osteosarcoma cells (Georgakis et al., 2006; Gazitt et al., 2009). Although the inhibitory role of 17-AAG in HCC cell has been investigated, its role in regulating cyclin D1 expression has not been reported. We found that 17-DMAG significantly down-regulated cyclin D1 protein level in HCC cells. Furthermore, over 50% of human tumours retain WT (wild-type) p53, but its function is defective due to abnormalities in p53 regulation. The MDM2 (murine double minute-2) and MDMX (murine double minute-X) proteins are the main p53 repressors, which mediate p53 degradation and transcriptional repression (Goldstein et al., 2011). The HSP90 inhibitor 17-AAG has been demonstrated to induce robust MDMX degradation, thereby increasing p53 transcriptional activity (Vaseva et al., 2011). In this study, 17-DMAG significantly increased p53 protein level in HCC cells. Moreover, HSP90 was reported to mostly promote cell survival through its involvement in the formation of active NF-κB. For example, HSP90 and its co-chaperone cdc37 are involved in the formation of active IKK [inhibitory κB (IκB) kinase] or Akt [also known as PKB (protein kinase B)] complexes, which can then phosphorylate IκB and thereby cause disassociation of NF-κB from its inhibitor and relocation into a cell's nucleus (Chen et al., 2002). In this study, 17- DMAG not only inhibited HSP90 but also inhibited NF-κB protein level as a result of HSP90 inhibition.

HCC is a multi-factor synergistic process that includes viral infection, oncogene activation and inactivation of tumour suppressor genes (Tommasi et al., 2007). A promising therapeutic strategy for treating cancer should specifically target a core molecule of multiple signalling pathways, thereby destroying the tumour cell survival network (Kaelin, 1999). HSP90 is an important molecular chaperone and regulates multiple signalling pathways involved in invasion, angiogenesis and metastasis of tumours (Stingl et al., 2010). In this study, 17-DMAG inhibited HCC cell proliferation and induced apoptosis through regulating survivin, cyclin D1, p53 and NF-κB protein levels. This suggests that HSP90 is a key core molecule of these signalling pathways (Figure 4C). Therefore the mechanisms of how 17-DMAG mediated the inhibition of cell proliferation and apoptosis in HCC cells could be proposed as follows: first, the binding of 17-DMAG to HSP90/survivin complex causes degradation of survivin that results in subsequent cell apoptosis because survivin is a strong inhibitor of apoptosis (Fortugno et al., 2003a, 2003b). Secondly, the binding of 17-DMAG to Akt or IKK reduces the frequency of NF-κB relocation to the nucleus. Otaki et al. (2000) found that the promoter region of the survivin gene also contains the binding sites of NF-κB and binding of NF-κB to survivin stimulates its expression. Therefore the reduced frequency of NF-κB relocation will cause a decrease in survivin expression. Thirdly, 17-DMAG may also degrade MDM2 or MDMX (Vaseva et al., 2011), and subsequently up-regulate p53 protein level. The activated p53 can induce apoptosis. Fourthly, the promoter region of survivin contains two p53 binding sites and the binding of p53 down-regulates survivin protein level (Sah et al., 2006). Interestingly, survivin is expressed in the G2/M phase of the cell cycle in a cycle-regulated manner (Ambrosini et al., 1997), while inhibition of survivin reduces cell proliferation by down-regulating cyclin D1 expression (Ai et al., 2006). Thus, the down-regulated cyclin D1 expression might be the cause of the down-regulated survivin level. Therefore it is not surprising that HSP90 is an emerging therapeutic target of interest for the treatment of HCC.

In conclusion, inhibition of the molecular chaperone function of HSP90 significantly inhibited the growth of HCC cells. HSP90 inhibitor 17-DMAG inhibited cell proliferation and induced apoptosis through the direct degradation of survivin, up-regulation of p53 through degradation of p53 inhibitors, reduction of NF-κB relocation and cyclin D1 expression. The 17-DMAG treatment of HCC provides a promising strategy for the anti-tumour therapy of HCC and a tool for exploring the complex mechanism of HSP90 in primary liver cancer.

Author contribution

Ai-min Leng and Ting Liu designed and performed the experiments, and wrote the paper. Jing Yang, Jian-fang Cui and Xiu-hua Li performed experiments. Ya-nan Zhu and Ting Xiong performed data analysis. Guiying Zhang and Yuxiang Chen supervised the study, revised the paper, and gave final approval of the paper.

Funding

This work was supported by the Chinese National Natural Science Foundation [grant number 30800518] and the New Teachers of Doctor Fund Project of Ministry of Education [grant number 200805331090].

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Received 26 August 2011/14 November 2011; accepted 14 June 2012

Published as Cell Biology International Immediate Publication 14 June 2012, doi:10.1042/CBI20110473


© The Author(s) Journal compilation © 2012 International Federation for Cell Biology


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)