Background
Ovarian cancer is the most lethal gynecological malignancy [
1], which seriously threatens the health of women. Current treatment for ovarian cancer includes a combination of surgery and chemotherapy [
2]. However, many patients carry poor prognosis even after long-term systemic therapy with cytotoxic agents [
3]. Ever since the platinum-based chemotherapy was applied to clinic, the survival rate of women with ovarian cancer has had no obvious improvement for nearly 40 years, thus, there is a need to develop more effective agents to improve this situation.
Temozolomide-perillyl alcohol conjugate (NEO212), a novel temozolomide (TMZ) analog, is developed based on the conjugation of TMZ, a clinically applied alkylating agent, and perillyl alcohol (POH), a naturally occurring monoterpene that has been used orally for the treatment of a variety of cancers [
4]. This novel compound has recently attracted continuous attention since accumulating data demonstrate that it exhibits stronger anti-cancer potency than either of its individual components (TMZ and POH) in several types of malignancy such as glioma [
5], triple-negative breast cancer (TNBC) [
6], non-small cell lung cancer (NSCLC) [
7,
8] and human nasopharyngeal carcinoma (NPC) [
9]. In this paper, we intend to illuminate the potential anticancer property of NEO212 in ovarian cancer cells.
Mitochondria as crucial organelles regulate cellular energy generation, calcium and redox homeostasis and apoptosis. Given that, targeting mitochondria in cancer cells is considered as an attractive therapeutic strategy [
10]. To perform the cellular functions effectively, mitochondria continuously change their structure and morphology through protein machineries controlling fission and fusion process [
11]. Fission is needed to create new mitochondria, but it can also facilitate apoptosis during high levels of cellular stress [
12] such as chemical reagent treatment. This occurs almost simultaneously with two steps of apoptosis that involve mitochondria: translocation from the cytosol to mitochondria of the pro-apoptotic Bcl-2 family member BAX and Cytochrome C release [
13]. Our previous studies have demonstrated that NEO212 was capable of inducing ROS accumulation, mitochondrial membrane potential collapse and mitochondrial apoptosis [
7,
9], indicating mitochondria might be one of main targets of NEO212.
Damaged mitochondria can trigger apoptosis, it prevents cellular harm and is crucial for cell survival. An increasing number of researches shows that autophagy can give cancer cells resistant ability to apoptosis, whereas inhibition of autophagy causes apoptotic cell death [
14] [
15]. Understanding the crosstalk between apoptosis and autophagy in cancer cells is vital to find out new strategies for cancer therapy and improve the therapeutic efficiency. Hence, the ability of NEO212 to affect apoptosis and autophagic activity in ovarian cancer cells should be revealed. Previous studies have established the association between chemotherapy with TMZ and autophagy [
16]. Interestingly, when autophagy was prevented at an early stage by 3-methyladenine (3MA), the antitumor effect of TMZ was suppressed, whereas bafilomycin A1 (Baf.A1) that prevents autophagy at a late stage by inhibiting lysosome acidification and its fusion with autophagosome, sensitized tumor cells to TMZ by inducing apoptosis, indicating the chemotherapy efficacy of TMZ depends on it induced autophagic flux status.
Terminally damaged mitochondria are sequestered in a double membrane vesicle (autophagosome), and then fuses with a lysosome, causing its contents to be degraded. The function of autophagosome and lysosome can be regulated by some transcriptional factors, such as transcription factor EB (TFEB). TFEB regulates lysosomal biogenesis by stimulating the expression of proteins involved in all steps of lysosome [
17], is activated by an increased need for lysosomal activity or when lysosomal function is impaired [
18]. Interestingly, TFEB is also involved in mitochondrial dysfunction and has been reported to regulate mitochondrial biogenesis via inducing the expression of major related master regulator genes [
19], indicating that mitochondria and lysosomes share strong functional links that could play a fundamental role in both normal physiology and pathology [
20].
In the current study, we aimed to reveal the potential anticancer property of NEO212 in ovarian cancer and the underlying mechanisms. We found that NEO212 exerted an antitumor effect in ovarian cancer, as evidence from cell proliferation inhibition, G2/M arrest, DNA damage, xenograft, mitochondrial fission and apoptosis. Importantly, we observed that NEO212 blocked autophagy flux although the number of autophagosomes was increased, which significantly facilitated it induced apoptosis and was largely because NEO212 inhibited the nuclear translocation of transcription factor EB (EB), and impaired the lysosomal function, thus proposing NEO212 as a potential therapeutic candidate for ovarian cancer.
Methods and materials
Cell lines and chemicals
Human ovarian cancer-derived cell lines SK-O-V3 and OVCAR-3 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA); A2780 were purchased from KeyGEN BioTECH (Jiangsu, China). A2780 and OVCAR-3 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Thermofisher, Carlsbad, CA, USA), SK-O-V3 cells were grown in McCoy’s 5A (modified) Medium (Thermofisher), supplemented with 10% fetal bovine serum (Thermofisher) and antibiotics (penicillin/streptomycin, 100 U/ml, Beyotime, Beijing, China) at 37 °C in 5% CO2.
NEO212 and Perillyl alcohol (POH) were provided by Neonc Technologies, Inc. (Los Angeles, USA) and diluted with DMSO to make stock solutions of 100 mM. Temozolomide (TMZ), 3-methyladenine (3MA), Baflomycin A1 (Baf.A1), Earle’s Balanced Salt Solution (EBSS) (Sigma-Aldrich, Shanghai, China) were dissolved in DMSO or deionized water dependently; In all cases of cell treatment, the final DMSO concentration in the culture medium never exceeded 0.5%. Stock solutions of all drugs were stored at − 20 °C.
Adenoviral infection
Recombinant adenoviral vector carrying the human mRFP-GFP-LC3 gene was purchased fom HanBio (Wuhan, China). Cells were plated in 12-well plates at a density of 1 × 104 cells per well, and infected at a MOI of 2 with mRFP-GFP- LC3 gene for 24 h. After washing with PBS twice, cells were treated with NEO212 for another 48 respectively.
Detection of apoptotic cells
Apoptosis was evaluated by using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences Pharmingen, San Diego, USA) according to the description provided by the manufacturer. After drug treatment, the cells were trypsinized, collected, washed once with PBS and stained with FITC-Annexin V & Propidum Iodide (PI) for 15 min in the dark. The stained cell population were determined using a FACS Calibur instrument (Becton Dickinson, Bedford, MA, USA) and the data were analyzed using FlowJo Software 7.6 (Treestar, Inc., San Carlos, CA). In some experiments, a pan-caspase inhibitor Z-VAD-FMK (20 μM, Z-VAD, KeyGEN BioTECH), and a specific caspase-9 peptide inhibitor Ac-LEHD-FMK (10 μM, Ac-LEHD, KeyGEN BioTECH), were employed along with or without NEO212.
Cell cycle analysis
Cells growing in 6-well plates were treated by above agents, cells were collected and washed once with PBS, and then re-suspended and fixed in 70% ethanol overnight. After incubation in 1 ml of propidium iodide staining solution (0.1% Triton X-100, 200 μg/ml DNase-free RNase A, 20 μg/ml propidium iodide) for 1 h at room temperature, DNA content was evaluated by a FACS Calibur instrument (Becton Dickinson, USA) and the distribution of cell cycle phases was determined using ModiFit software (Topsham, ME, USA).
Depending on the cell line, 100–350 cells were seeded into each well of a 6-well plate and exposed to 100 μM TMZ, POH, TMZ + POH and NEO212 or DMSO for 48 h, respectively, then drugs were withdrawn and cells grew in normal culture for 14 days, DMSO acted as the control, and subjected to cell colony formation assay. After fixation with acetic acid–methanol (1:4) and staining with diluted crystal violet (1:30), colonies that consisted of > 50 cells were counted and calculated. The colony formation efficiency was calculated with the following formula: Survival Fraction = Clones/Cell numbers × 100%. Three independent experiments were carried out.
Transmission electron microscope (TEM)
Cells were fixed in TEM stationary solution (2.5% glutaraldehyde in 0.2 M HEPES, G1102, Servicebio Technology) at 4 °C for 4 h, rinsed in PBS, and then embedded in 4% agarose. After fixation in 1% osmium tetroxide for 2 h, the specimens were dehydrated using alcohol and embedded in polybed 812 resin (90529–77-4, SPI). After polymerization at 60 °C for 48 h, ultrathin sections were prepared with the Leica Ultracutuct slicer (Leica EM UC6, Germany), stained with uranyl acetate and lead citrate, and analyzed using TEM (HT7700, HITACHI). Count, measure and analysis on TEM picture were carried out using Fiji ImageJ software.
Analysis of mitochondrial transmembrane potential (Δψm)
Cancer cells grown in six-well plates overnight were exposed to indicated drugs, then stained with JC-1 (Beyotime, Beijing, China) to demonstrate the state of mitochondrial transmembrane potential according to the manufacturer’s protocol. Briefly, cells were harvested and transferred to 1.5 ml tubes, and then incubated with JC-1 (5 μg/ml) in a 37 °C incubator for 20 min after washing twice with PBS. Subsequently, cells were collected and subjected to flow cytometry (Becton Dickinson) to detect the change of JC-1 florescence. The data were analyzed using FlowJo Software 7.6 (Treestar).
Cytoplasmic and nuclear extraction
pEnter vector carrying Flag tagged ORF of TFEB (Flag-TFEB) was purchased from Vigene Bioscience (Shandong, China). A2780 and SK-O-V3 cells were seeded into a 6-well plate, incubated overnight and then transfected with pEnter Flag-TFEB using Lipofectamine 3000 (Thermofisher) according to the manufacturer’s instructions to selectively upregulate Flag-TFEB. After 24 h, cells were treated with or without NEO212 in normal medium for 32 h plus EBSS for another16 h, then were subjected to cytoplasmic and nuclear extraction.
Cytoplasmic and nuclear extraction was performed using MINUTE™ CYTOPLASMIC AND NUCLEAR KIT (Invent, Beijing, China) according to the manufacturer’s instructions. Briefly, cytoplasmic extraction buffer was added to cells after twice washing with cold PBS and lysed on ice for 5 min; then lysate was centrifuged; supernatant was transferred (cytosol fraction) to a fresh pre-chilled 1.5 ml tube, whereas pellet was added with nuclear extraction buffer, vortexed vigorously and then transferred to a pre-chilled filter cartridge (nuclear fraction). Pre-chilled filter cartridges were centrifuged at 16000 g for 30 s. The filter cartridges were discarded, and protein was collected.
Immunofluorescence (IF) staining
For immunostaining, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 15 min. After incubation for 1 h with the following primary antibodies: anti-LC3B (1:300, Cell Signaling Technology, Danvers, MA, USA), anti-TFEB (1:300, Proteintech, PTG, China) and washing with PBS, cells were incubated for 1 h with Alexa 488-conjugated (1:1000) (1:500, Abcam, Shanghai, China) secondary antibodies, washed with PBS. Nuclei were stained by 4′, 6-diamidino-2-phenylindole (DAPI) (Beyotime) for 3 min. Microscopy was done on a confocal laser microscopy (LSM800, Carle Zeiss, Germany).
For quantification of the number of autophagosomes, at least five cells were randomly chosen, all eligible puncta were recorded and analyzed using Fiji ImageJ software [
21]. Quantification of GFP and mRFP fluorescence intensity, and colocalization between two different signals were recorded and analyzed using Fiji ImageJ software.
Fluorescence probe detection
Cells following the above treatment were loaded fluorescence probe Mito-Tracker Green (MTG, Beyotime) for 20 min. After washing 3 times with PBS, the florescence intensities were observed under a confocal laser microscopy (LSM800).
Western blots
Cells were lysed in Cell Lysis Buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100) (Beyotime) supplemented with 0.5 mM phenylmethanesulfonyl fluoride (PMSF, Beyotime), and the total cellular protein concentration was determined with a BCA Protein Assay Kit (Thermofisher). 50 μg quantity of protein was separated on SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Membranes were then blocked with 5% evaporated skimmed milk (Bio-rad, USA) in Tris-buffered saline (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.1% Tween-20 for 1 h, and probed overnight at 4 °C with the following primary antibodies: antibodies against human LC3B, BECN1, SQSTM1, pho-ATM, γ-H2AX, pho-CHEK1/2, Cyto C, BAX, cl-CASP3, CASP3, cl-CASP9, AKT, pho-AKT, pho-ERK (1:1000; CST), EEA1, LAMP1, LAMP2, TFEB, Lamin B1, GAPDH, ACTB (1:1000; PTG) followed by incubation with horseradish peroxidase coupled secondary anti-mouse or anti-rabbit antibodies (1:2000; PTG) for 1 h at room temperature. The protein bands were visualized using ECL blotting detection reagents (Bio-rad, USA), and developed and fixed onto x-ray films. ACTB or GAPDH or Lamin B1 was served as a loading control dependently.
In vivo studies
BALB/c nude mice (4–6 weeks of age, female) were purchased from Beijing HFK Bioscience Co., Ltd. (Beijing, China). Mice were housed and handled in laminar flow cabinets under specific pathogen-free conditions according to institutional guidelines and experimental procedures approved by the Institutional Animal Care and Use Committee of Shandong Cancer Hospital and Institute with full respect to the EU Directive 2010/63/EU for animal experimentation. Approximately 5 × 106 SK-O-V3 cells in 100 μl PBS were innoculated s.c. into the left flank of nude mice. When reached approximately 100 mm3 in size 1 week later, mice were randomly divided into five groups, and treated once a day for 15 days as follows: DMSO, TMZ, POH, TMZ + POH and NEO212. Size of local tumors and mouse body weight were calculated by measuring two perpendicular diameters (length and width) every two days using a caliper, and the volume was calculated according to the formula: tumor volume (mm3) = 1/2 × (length × square width). The mice were sacrificed after completion of treatment and the tumors were separated and weighted.
Immunohistochemical (IHC) staining
The xenograft tumor tissues were fixed and paraffin-embedded for section. After routinely dewaxing and hydration, antigen in specimens proceeded to be repaired, the slides were blocked for endogenous peroxidase activity, preincubated with goat serum, and then stained with anti-cl-CASP3 (Servicebio Technology)) for 1 h at room temperature. Secondary staining was carried out with HRP- conjugated anti-rabbit IgG and DAB peroxidase substrate.
Hematoxylin-eosin (H&E) staining
The slides were deparaffinized then stained with hematoxylin (2 min), rinsed with distilled water, rinsed with 0.1% hydrochloric acid in 50% ethanol, rinsed with tap water for 15 min, stained with eosin for 1 min, and rinsed again with distilled water. The slides were then dehydrated and mounted with coverslips.
Statistical analysis
Statistical significance was evaluated with data from at least two independent experiments or at least five duplicates. GraphPad Prism 6.02 (GraphPad Software, San Diego, CA, USA) was used for data analysis. Statistical analysis was carried out using Student t-test for two groups as well as one-way ANNOVA for more than two groups. Data are presented as the mean ± SD. For all statistical tests, significance was established at P < 0.05.
Discussion
In current study, we provide a novel report on the cytotoxic effect of NEO212 on ovarian cancer in vitro and in vivo, and the underlying mechanisms. TMZ, a novel alkylating agent, had insignificant activity in conspicuous therapeutic effect for ovarian cancer [
32], whereas the newly designed compound NEO212, a novel temozolomide analog, exerted stronger cytotoxicity than its individual constituents, as evidence from stronger inhibition of cell proliferation and colony formation, along with higher level of G
2M arrest, DNA damage, mitochondrial dysfunction and apoptosis, thereby proposing NEO212 as a potential candidate for ovarian cancer therapy.
Here, we believed that mitochondria were one of main sites that NEO212 exerted its cytotoxicity. We found that NEO212 damaged the mitochondrial morphology and activity, as proved by increased fission, destroyed shape and transmembrane potential collapse when treated by NEO212 in ovarian cancer, similar to those in NSCLC [
7] and NPC [
9] cells. Diversely, in NSCLC and NPC cells, NEO212 induced ROS accumulation was the key contributor to its cytotoxicity because it can be reversed by two ROS scavengers, catalase (CAT) and N-acetyl-L-cysteine (NAC); However, NEO212 seemed not alter the level of intracellular ROS accumulation (data not shown), indicating alternative pathway was involved in. We supposed NEO212 as an alkylating agent might works due to its capability to methylate mitochondrial DNA directly and induce its dysfunction, although this hypothesis needs further investigation.
Importantly, our data supported that NEO212 induced apoptosis via mitochondrial pathway. This is because NEO212 was capable to regulate a series of mitochondrial apoptosis related genes, such as BAX, Cyto C and cl-CASP9. Cyto C, a pro-apoptotic factor, releases on the outer surface of the inner mitochondrial membrane at early steps of apoptosis which can be initiated by the pro-apoptotic protein BAX, combining with some cytosolic proteins, activates conversion of the latent apoptosis-promoting protease pro-CASP9 to its active form, and finally mitochondrial apoptosis. This conclusion was supported by a pan CASP inhibitor and a specific CASP9 inhibitor analysis, both of which succeeded in relieving NEO212 induced apoptosis, indicating a caspase dependent manner involved. Notably, in our previous study, the pan caspase inhibitor failed to prevent cells from NEO212 cytotoxicity in NSCLC cells [
7], which might attribute to the cell specificity.
Dysfunctional and damaged mitochondria initiate the protective mechanisms, such as autophagy, a conserved eukaryotic catabolic reaction for removal and recycling, thereby it has been proposed as a mechanism of chemoresistance to alkylating drugs [
33]. Depending on the cellular context, autophagy may lead to tumor cell survival or death. Inhibition of autophagy by 3MA failed to increase TMZ sensitivity as observed with Baf.A1 but, instead, enhanced TMZ chemoresistance of glioma cells [
16], since TMZ induced an autophagy-associated increase in ATP production, which was blocked by pre-incubation with autophagy inhibitor 3MA, and increased non-apoptotic cell death [
34]. In current study, we also explored the association of NEO212 induced autophagosome accumulation and apoptosis. 3MA appeared no any positive or negative effect on NEO212 induced apoptosis, whereas Baf.A1 that prevents autophagy at a late stage sensitized tumor cells to NEO212 by inducing apoptosis, indicating NEO212 might interrupt autophagy flux. Lysosomotropic agents, like Baf. A1 and chloroquine (CQ), impair lysosome function and its fusion with autophagosome and inhibit autophagic flux, and in turn eliminate the protective role of autophagy, which have been used successfully to overcome chemoresistance against alkylating drugs. In current study, we observed that NEO212 quenched GFP-LC3 degradation, down-regulated a series of lysosome related gene expression, and blocked the autophagic flux, although it induced significant accumulation of autophagosomes, implying NEO212 might avoid from autophagy-mediated chemoresistance.
Collectively, we add a novel insight into the role and underlying mechanisms of NEO212 in ovarian cancer. We have demonstrated NEO212 exerted stronger cytotoxicity than its individual constituents, as evidence from stronger inhibition of cell proliferation and colony formation, along with higher level of G2M arrest, DNA damage, mitochondrial dysfuncton and apoptosis. Besides, NEO212 was capable to down-regulate a series of lysosome related gene expression via hindering the nuclear translocation of TFEB, and in turn block the autophagic flux, implying NEO212 might as a promising therapeutic agent against ovarian cancer.
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