Platinum‐based combination chemotherapy triggers cancer cell death through induction of BNIP3 and ROS, but not autophagy

Abstract These days, cancer can still not be effectively cured because cancer cells readily develop resistance to anticancer drugs. Therefore, an effective combination of drugs with different mechanisms to prevent drug resistance has become a very important issue. Furthermore, the BH3‐only protein BNIP3 is involved in both apoptotic and autophagic cell death. In this study, lung cancer cells were treated with a chemotherapy drug alone or in combination to identify the role of BNIP3 and autophagy in combination chemotherapy for treating cancer. Our data revealed that various combinational treatments of two drugs could increase cancer cell death and cisplatin in combination with rapamycin or LBH589, which triggered the cell cycle arrest at the S phase. Cells with autophagosome and pEGFP‐LC3 puncta increased when treated with drugs. To confirm the role of autophagy, cancer cells were pre‐treated with the autophagy inhibitor 3‐methyladenine (3‐MA). 3‐MA sensitized cancer cells to chemotherapy drug treatments. These results suggest that autophagy may be responsible for cell survival in combination chemotherapy for lung cancer. Moreover, BNIP3 was induced and localized in mitochondria when cells were treated with drugs. The transfection of a dominant negative transmembrane deletion construct of BNIP3 (BNIP3ΔTM) and treatment of a reactive oxygen species (ROS) inhibitor suppressed chemo drug‐induced cell death. These results indicate that BNIP3 and ROS may be involved in combination chemo drug‐induced cell death. However, chemo drug‐induced autophagy may protect cancer cells from drug cytotoxicity. As a result, inhibiting autophagy may improve the effects of combination chemotherapy when treating lung cancer.


| INTRODUC TI ON
Lung cancer is the leading cause of cancer-associated deaths worldwide. Two major clinical types of lung cancer have been well identified, which are small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). Approximately 85% of lung cancer is histologically classified as NSCLC. The 5-year survival rate of most patients with metastatic NSCLC remains low. 1 In general, two chemotherapy drugs combined are standard for treating NSCLC. Elderly patients or those in poor health may not tolerate combination chemotherapy and may thus be given single-drug chemotherapy. Combination chemotherapy frequently contains cisplatin or carboplatin plus pemetrexed, gemcitabine or docetaxel. For patients with advanced lung cancers, a targeted therapy or immunotherapy drug such as bevacizumab, erlotinib or pembrolizumab may be added to the treatment. 1 However, most patients acquire drug resistance at around 6-18 months, so continued research on new combination therapies and drug resistance mechanisms can improve the outcomes of NSCLC patients.
Cisplatin (CDDP), which belongs to a class of platinum-containing antineoplastic drugs, is the first-line chemotherapy regimen for many kinds of cancers. Cisplatin triggers cell apoptosis by binding to DNA and crosslinking the DNA strands. However, its efficacy is limited due to adverse drug effects and the development of drug resistance. Several strategies can overcome cisplatin drug resistance and toxicity, including liposome delivery, inhibition of glutathione and metallothionein species, and combination therapy. 2 The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that controls cell proliferation and survival. AKT1 overexpression and gene amplification have been shown to induce lung cancer cells to become resistant to cisplatin through mTOR signaling pathway. 3 Rapamycin, also known as sirolimus, is a well-studied mTOR inhibitor and has potent immunosuppressive and antiproliferative properties.
It is a promising therapeutic agent for solid organ transplantations and cancer treatments. 4 In addition to the direct antiproliferative effect on tumour cells, rapamycin impedes tumour growth by blocking angiogenesis in a mouse metastatic cancer model. 5 LBH589 (panobinostat), a non-selective histone deacetylase inhibitor (pan-HDAC inhibitor), has been shown to induce tumour shrinkage and sustain stable disease status in a phase II trial of pre-treated lung cancer patients. 6 LBH589 is the first HDAC inhibitor to be approved by the FDA to treat multiple myeloma patients. 7 Autophagy is a self-digesting catabolic process induced under various stress conditions for homoeostatic cellular recycling that has been implicated in the progression of many diseases, including cancer and neurodegeneration. 8 Autophagy has been found to play a paradoxical role in anticancer treatments. In some pharmacological studies, autophagy induction is required for immunogenic cancer cell death. On the other hand, activated autophagy mediates drug resistance and survival in some cancer cells. 8,9 BNIP3 (Bcl-2/adenovirus E1B 19 kD protein-interacting protein 3) is a pro-cell death member of the Bcl-2 family and contains only the BH3 domain. BNIP3 forms stable homodimerization complexes on the outer membrane of mitochondria to induce apoptosis, necrosis and autophagy after cellular stress. 10 The carboxy terminal tail of BNIP3, but not BH3, is essential for inducing mitochondrial permeability transition and cytochrome c release, while the transmembrane (TM) domain is required for mitochondrial localization. 11 BNIP3 expression is down-regulated by promoter hypermethylation or homozygous gene deletion in certain cancers, whereas their expression increased and were correlated with poor prognosis in other cancers. 10 Furthermore, the lack of BNIP3 expression has been linked to chemoresistance of pancreatic cancer cells to gemcitabine and 5-fluorouracil. 12,13 However, the role of BNIP3 regarding cell death in the combination chemotherapy of lung cancer is not yet properly understood. In this study, we used cisplatin and two novel chemo drugs (LBH589 and Rapamycin) with different molecular mechanisms to treat lung cancer cells. We found that BNIP3 and ROS may be involved in combination chemo drug-induced cell death and inhibition of autophagy to improve the effects of combination chemotherapy in treating lung cancer.
After 30 minutes of incubation, cells were washed twice with PBS.
Decanting of all the supernatant was followed by adding 800 μL PBS, 100 μL propidium iodide (400 μg/mL) and 100 μL RNase A (1 mg/mL) to the sample. The cells were incubated for 30 minutes at 37°C in the dark and then analysed using FACS Calibur (BD Biosciences).

| Reverse transcription and quantitative polymerase chain reaction (RT-qPCR)
Total RNA extraction from A549 cells was performed with TRIzol

| Western blotting
Cells were treated with the drugs cisplatin, LBH589 and rapamycin, either alone or in combination. Cell lysates were extracted into RIPA Lysis buffer (Millipore). Protein concentrations were measured, and equal amounts of total protein were resolved using a 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membrane (Millipore). The membrane was blocked in 5% skim milk for 1 hour and incubated with the indicated antibodies and horseradish peroxidase-conjugated secondary antibodies. The immunocomplexes were detected using the chemiluminescence HRP substrate (ECL, Millipore Corporation).

| Construction of pEGFP-BNIP3 and ΔTM-BNIP3 plasmids
Cloning of BNIP3 and deletion mutant was carried out as described in a previous study. 11 Briefly, full-length DNA encoding BNIP3 was amplified by RT-PCR from human embryonic kidney (HEK) 293 cells and cloned into pEGFP (Clontech Laboratories, Inc). Using the splice overlap extension method, 11 the transmembrane deletion mutant (BnipΔ164-183 and ΔTM-BNIP3) was cloned into pcDNA3 (Invitrogen). Sequences of BNIP3 and the deletion mutant were confirmed by Mission Biotech Co.

| Transient transfection
A549 cells were seeded 18 hours prior to transfection with plasmids using jetPEI™ (Polyplus Transfection) transfection reagent according to the manufacturer's instruction. In short, the plasmid and jetPEI™ mixture was added to cells and incubated for 24 hours. Afterwards, the medium was replaced with a fresh, complete medium. For pEGFP-LC3 transfection, cells were observed under fluorescence microscopy (Leica DM 6000B).  (1 μg/mL) at 37°C for 20 minutes. After washing three times with PBS, cells were trypsinized with trypsin-EDTA, suspended in phenol red-free RPMI medium and then analysed immediately using flow cytometry.

| Statistical analysis
Data are shown as mean ± standard deviation of three independent experiments, and statistical significance was assessed by Student's t test. Differences with a P-value of < .05 were considered to be statistically significant.

| Combination of two anticancer drugs with different mechanisms induced cancer cell death more severely
First, to clarify whether a combination of anticancer drugs with different mechanisms could induce higher cell death of lung cancer cells, we used a DNA damaging agent (cisplatin (CDDP), HDAC inhibitor (LBH589) and mTOR inhibitor (rapamycin). A549 cells were treated with these drugs alone or in combination, and then their cell viability was measured. As shown in Figure 1A and Table 1

| The induction and mitochondrial localization of BNIP3 were identified upon anticancer drug treatment
AsBNIP3 has been reported as an important molecule in mitochondrial autophagy and cell death, 10  analysis clearly showed that the expression of BNIP3 was dramatically up-regulated in the chemo drug combination regimen, especially in cisplatin-treated groups, which was co-localized with mitochondria.
These findings indicated that anticancer drug-induced BNIP3 localized in mitochondria ( Figure 2C).

| Chemotherapy drugs induced autophagy in lung cancer cells
To illustrate whether anticancer drugs can induce autophagy, we analysed the autophagy-related genes by RT-qPCR. As expected,  Figure 4D). These data indicated that anticancer drugs were effective in inducing the autophagy of lung cancer cells.

| Autophagic inhibitor 3-MA suppressed chemotherapeutic drug-induced autophagy
To determine whether chemo drug-induced autophagy can be inhib-

| Inhibition of autophagy augments chemotherapeutic drug-induced cell death
To investigate whether chemotherapy drug-induced autophagy leads to cell survival or cell death, we detected the viability of cells pretreated with or without 3-MA and chemotherapy drugs. We found that

| D ISCUSS I ON
Poor prognosis of NSCLC is mainly due to initial diagnosis occurring at an advanced stage. Despite progress in new therapies, truly effective therapy is still vital for improving the survival of NSCLC patients. 1 Combination therapy targeting different molecular key pathways is usually selected to enhance therapeutic efficacy and reduce drug resistance of cancer patients in clinics. 15 Pathways involved in tumour growth that can be targeted include antioxidants, growth factors, angiogenesis, epigenetic modification and signalling pathway.
Platinum-based doublet cytotoxic therapy is the standard therapy for advanced-stage NSCLC patients. However, only cisplatin combined with pemetrexed showed significant differences in clinical outcome among the multiple cytotoxic treatments for advanced NSCLC patients. 1,16 In this study, we combined cisplatin with other new pathway inhibitors, mTOR and HDAC inhibitors, to treat NSCLC cancer cells.
We found that cisplatin (CDDP) plus HDACi (LBH589) had a greater synergistic effect than cisplatin plus mTOR inhibitor (rapamycin) on cell growth inhibition. Furthermore, LBH589 combined with rapamycin had less effect on cancer cell growth.
Epigenetic regulations of gene expression, including DNA methylation, histone acetylation and methylation, non-coding been explored in phase II/III clinical trials. 18  Autophagy plays a pro-tumoural or anti-tumoural role in different stages for cancer progression. 8  to activate autophagy. Therefore, levels of the BNIP3 C-terminus phosphorylation control the pro-survival and pro-death activities of BNIP3 in response to extracellular stress. 24 In the present study, we found that the C-terminal TM domain of BNIP3 was involved in chemo drug-induced cell death ( Figure 3). Nevertheless, the role of Hospital, Taiwan.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest in relation to this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.

R E FE R E N C E S F I G U R E 7
A proposed model for the role of BNIP3 and autophagy in chemotherapeutic drug-induced cell death. Anticancer drugs augment Binp3 and autophagy in lung cancer cells. Anticancer drugs induction of mitochondrial membrane insertion of BNIP3 may promote ROS and potential membrane loss, resulting in apoptotic or necrotic cell death. However, anticancer drug-induced autophagy may also be involved in cell survival