J Lung Cancer.  2010 Dec;9(2):77-84. 10.6058/jlc.2010.9.2.77.

Combination Therapy of Bortezomib (PS-341) and SAHA (Vorinostat) in Non-Small Cell Lung Cancer Cell Lines

• Park MY
• Kim DR
• Park JS
• Cho YJ
• Lee SW
• Yoon HI
• Lee JH
• Lee CT

• KMID: 2199908
• DOI: http://doi.org/10.6058/jlc.2010.9.2.77

Abstract

PURPOSE
The recent introduction of targeted therapy for non-small cell lung cancer (NSCLC) has changed the paradigm of lung cancer chemotherapy. However, only a small portion of NSCLC patients received the benefit of these new drugs. A proteasome inhibitor (bortezomib) and a histone deacetylase inhibitor (SAHA) were approved for clinical use for treating some hematologic malignancies. In this study, we investigated the combination treatment of bortezomib and SAHA in NSCLC cell lines.
MATERIALS AND METHODS
The combined effects of bortezomib and SAHA on lung cancer cell lines were measured by Calcusyn software. Induction of apoptosis was examined by performing an Annexin V assay. Generation of reactive oxygen species (ROS) and protection by N-acetylcysteine were measured by flow cytometry after staining with DCFH-DA. The effect of the combination of drugs on apoptosis and autophagy was investigated by Western blot assay.
RESULTS
Strong synergism was found for the combination of bortezomib and SAHA. The synergistic interaction was mediated by strong apoptosis. Increased ROS generation was partly responsible for the induction of apoptosis, and this was suppressed by the ROS scavenger N-acetylcysteine. Combined treatment induced the strong activation of caspase-3 and the breakdown of the antiapoptotic molecule Bcl-2. Furthermore, increased breakdown of beclin-1, which is known to be an autophagic molecule, was also found.
CONCLUSION
Combination therapy with bortezomib and SAHA showed a strong synergistic antitumor effect on human lung cancer cell lines. Enhanced induction of apoptosis was a responsible mechanism, and this was partly mediated by ROS generation. Further studies are warranted for determining the role of apoptosis and autophagy for this combination therapy.

Keyword

Lung neoplasms; Bortezomib; Histone deacetylase inhibitor; Apoptosis; Reactive oxygen species

MeSH Terms

Acetylcysteine
Annexin A5
Apoptosis
Autophagy
Blotting, Western
Boronic Acids
Carcinoma, Non-Small-Cell Lung
Caspase 3
Cell Line
Flow Cytometry
Fluoresceins
Hematologic Neoplasms
Histone Deacetylase Inhibitors
Humans
Lung Neoplasms
Proteasome Inhibitors
Pyrazines
Reactive Oxygen Species
Bortezomib
Acetylcysteine
Annexin A5
Boronic Acids
Caspase 3
Fluoresceins
Histone Deacetylase Inhibitors
Proteasome Inhibitors
Pyrazines
Reactive Oxygen Species

Figure

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  Fig. 1. Analysis of the drug interaction (SAHA and bortezomib) in the lung cancer cell lines. Calcusyn analysis (right column) demonstrated that SAHA and bortezomib showed strong synergistic cytotoxicity in two lung cancer cell lines (the combination indices for most drug combinations were below 0.7).

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  Fig. 2. Increased apoptosis by combining SAHA and bortezomib in a lung cancer cell line. (A) NCI H460 cells were treated with SAHA (0, 1μ M) and bortezomib (0, 0.03, and 0.1μ M) for 48 hours and Annexin V assay was performed to measure the cell apoptosis. The cells treated with SAHA (1μ M) and bortezomib (0.1μ M) showed a high proportion of early apoptotic cells (24.96%) compared with the cells treated with SAHA (1μ M) alone (2.6%) and the cells treated with bortezomib (0.1μ M) alone (6.6%). (B) Data of the proportions of cells showing early apoptosis (lower right quadrant, LR) in three independent experiments (B, bortezomib; S, SAHA). All the experiments showed the same tendency of increased apoptosis by a combination of SAHA and bortezomib.

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  Fig. 3. The apoptosis induced by SAHA and bortezomib was suppressed by N-acetylcysteine (NAc), which is a reactive oxygen species (ROS) scavenger. Pretreatment of lung cancer cells (A549 and NCI H460) with NAc (15 mM).

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  Fig. 4. Increased reactive oxygen species (ROS) formation by combination of SAHA and bortezomib and reduced ROS formation by N-acetylcysteine (NAc). ROS was stained with DCFH-DA and ROS value was measured by flow cytometry. Three hour-pretreatment with NAc significantly reduced intracellular ROS formation induced by SAHA and bortezomib. Mean values of ROS in A549: 224.3 (native), 393.5 (bortezomib: 0.03μ M), 325.5 (SAHA: 1μ M), 459.5 (bortezomib＋ SAHA) and 274.5 (bortezomib ＋SAHA＋NAc). Mean values of ROS in NCI H460: 502.6 (native), 727.4 (bortezomib: 0.03μM), 535.1 (SAHA: 1μM), 1042.1 (bortezomib＋ SAHA) and 745.8 (bortezomib＋ SAHA＋ NAc).

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  Fig. 5. Analysis of apoptotic and autophagic pathway of lung cancer cells (A549 and NCI H460) treated with bortezomib and SAHA. Compared with single treatment of bortezomib or SAHA, combined treatment of bortezomib and SAHA induced enhanced cleavage of caspase 3 and breakdown of antiapoptotic molecule, Bcl-2. Furthermore, combined treatment of bortezomib and SAHA induced breakdown of beclin-1 known as autophagic molecule. Details are described in the text.

Reference

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