Targeted and Functional Genomics Approaches to the Mechanism of Action of Lagunamide D, a Mitochondrial Cytotoxin from Marine Cyanobacteria

Abstract

Lagunamide D, a cyanobacterial cyclodepsipeptide, exhibits potent antiproliferative activity against HCT116 colorectal cancer cells (IC₅₀ 5.1 nM), used as a model system to probe the mechanism of action. Measurements of metabolic activity, mitochondrial membrane potential, caspase 3/7 activity and cell viability indicate the rapid action of lagunamide D on mitochondrial function and downstream cytotoxic effects in HCT116 cells. Lagunamide D preferentially targets the G₁ cell cycle population and arrests cells in G₂/M phase at high concentration (32 nM). RNA-Transcriptomics and subsequent Ingenuity Pathway Analysis identified networks related to mitochondrial functions. Lagunamide D induced mitochondrial network redistribution at 10 nM, suggesting a mechanism shared with the structurally related aurilide family, previously reported to target mitochondrial prohibitin 1 (PHB1). Knockdown and chemical inhibition of ATP1A1 sensitized the cells to lagunamide D, as also known for aurilide B. We interrogated potential mechanisms behind this synergistic effect between lagunamide D and ATP1A1 knockdown by using pharmacological inhibitors and extended the functional analysis to a global level by performing a chemogenomic screen with a siRNA library targeting the human druggable genome, revealing targets that modulate susceptibility to lagunamide D. In addition to mitochondrial targets, the screen revealed hits involved in the ubiquitin/proteasome pathway, suggesting lagunamide D might exert its effects by additionally affecting proteostasis. Our analysis illuminated cellular processes of lagunamide D that can be modulated in parallel to mitochondrial functions. The identification of potential synergistic drug combinations that can alleviate undesirable toxicity may open possibilities to resurrect this class of compounds for anticancer therapy.

Graphical Abstract

1. Introduction

Marine natural products are emerging as important contributors for drug discovery, as developmental hurdles related to supply, novelty, as well as target identification and mechanism of action (MOA) can increasingly be overcome.[1] Marine cyanobacteria have yielded important drug leads as they produce compounds with intriguing pharmacological activities, including unique mechanisms.[1,2] Lagunamide D (Figure 1A) is a macrocyclic depsipeptide that was discovered along with its acyl migration product lagunamide D’ from marine cyanobacteria from Florida.[3] It exhibited exquisite potency against A549 human lung adenocarcinoma cells in the low-nanomolar range. Several structurally related compounds have been reported from marine sources, including aurilides[4–6] and lagunamides A-C.[7–8] Although efforts were expended to pursue synthetic analogues with even better drug-like properties, the development of a drug candidate was hampered due to the toxicity identified in animal testing.[6] However, MOA studies and chemical-genetic interaction analyses may enable an in-depth understanding of the cellular activity and identify cellular processes that impact the activity, ultimately providing opportunities to reduce toxic effects. Specifically, the identification of synergistic drug combinations and susceptible cell types/populations could potentially cause sufficient therapeutic effects with a reduced dose and therefore improve the safety profiles of this compound family. We aimed to probe the MOA of lagunamide D and identify genetic or cellular vulnerabilities that could be exploited for rational combination therapy.

Figure 1.

Antiproliferative effects of lagunamide D against HCT116 cells. (A) Structure of lagunamide D. (B) Lagunamide D effect on metabolic activity in cells using MTT reduction assay. (C) Percent cell viability measured using trypan blue staining and cell count at different time points. (D) Effect of lagunamide D on mitochondrial membrane potential measured using MITO-ID^® kit at 1, 3, 12 and 24 h. Carbonyl Cyanide Chlorophenylhydrazone (CCCP) provided with the kit was used as the positive control. (E) Effect of lagunamide D on caspase 3/7 activity in HCT116 cells measured at 3, 6 and 12 h. Data are presented as mean ± SD (n = 3), relative to solvent control.

The protein target of aurilide has previously been identified to be a mitochondria inner membrane protein, prohibitin 1 (PHB1), while no detectable affinity to the homolog prohibitin 2 (PHB2) was found.[9] While the role of aurilide has been extensively explored[9], no detailed information has been available for analogs from the lagunamide family beyond their ability to induce apoptosis[3,10,11]. It has been shown that mitochondrial functions are highly important in intestinal epithelial cell homeostasis; intestinal epithelial cells are key players in intestinal diseases, including colorectal cancer (CRC).[12] Therefore, the current study was performed mainly using the CRC cell line HCT116. Based on the structural similarity, members of both compound families likely share the same protein target(s). However, the structural differences, especially at their peptide fragments, may still lead to distinct alterations in target engagement and cellular functions. Therefore, we embarked on an in-depth biological study of lagunamide D, potentially adding more value to this compound family as a pharmacological tool or even revive opportunities for therapeutic development.

2. Materials and Methods

2.1. Isolation

The dark-brown encrusting cyanobacterial tufts (DRTO 85) were collected from Loggerhead Key, Dry Tortugas, Florida in 2015 and were identified as a mixture of Dichothrix sp., Lyngbya sp. with red algae Ceramium sp. serving as scaffold, and Rivularia sp. Lagunamide D (2.58 mg, >95% purity) was isolated from 71.3 g of freeze-dried material as previously described.[3]

2.2. Cell culture

HCT116 human colorectal carcinoma cells and HeLa human cervical carcinoma cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin (Invitrogen) and maintained under a humidified environment with 5% CO₂ at 37 °C.

2.3. Metabolic activity and cell viability measurement

HCT116 cells were seeded at a density of 10,000 cells per well in 96-well plates. Wells with negative control (cells + medium + solvent control) and medium only (medium + solvent control) were also incorporated for determination of 100% viability and background absorbance level. After 24 h of incubation, the cells were treated with varying concentrations of lagunamide D. The cells were incubated for 3, 12, 24, 48 h (the corresponding hours of treatment are also noted in figure) and observed under a microscope for changes in morphology before the addition of the MTT reagent (Promega, Madison, WI, USA). Cellular metabolic activity was measured according to the manufacturer’s instructions and recorded on a SpectraMax M5 or a Flexstation3 (Molecular Devices, San Jose, CA, USA). IC₅₀ calculations were done by GraphPad Prism 6 based on triplicate experiments.

Trypan blue viability measurement was performed in HCT116 cells seeded at 8000/well in 96-well plates and left to acclimate overnight. Cells were treated in triplicate wells with lagunamide D at 3-fold dilutions from 100 nM to 1 nM final concentration or with vehicle control (DMSO) and left to incubate at 37 °C. At 3, 12, 24 and 48 h post treatment, cells from 3 separate wells were lifted from wells by 0.05% trypsin, resuspended in new media and counted using hemocytometer chamber and trypan blue dye (1:1 dilution with sample) to calculate % stained (dead) vs % non-stained (alive) cells in each well of the respective triplicate. Data was averaged for the three replicates and graphed using GraphPad Prism software.

To measure the effect of cell cycle arrest on lagunamide D susceptibility, cells were arrested at G₀/G₁ stage by starving for 24 h with low-serum medium (DMEM, 0.5% FBS, and 1% penicillin/streptomycin) before use. Cells were arrested at G₂/M phases by treating with 650 nM nocodazole for 24 h and harvested by mechanical shaking. Cell viability was measured after treating with lagunamide D for 12 h or 48 h, respectively.

2.4. Caspase 3/7 activity

HCT116 cells were seeded at a density of 5000 cells per well in solid white flat bottom 96-well plates. Wells with negative control (cells + medium + solvent control) and medium only (medium + solvent control) were also incorporated for determination of baseline activity and background luminescence level, respectively. After 24 h of incubation, the cells were treated with varying compound concentrations of lagunamide D as indicated. At the end of the 3, 6 and 12 h incubation period, 60 μL medium was removed. The caspase-Glo 3/7 reagent was prepared according to the manufacturer’s instruction (Promega) and 40 μL reagent was added to each well. After incubation at room temperature for 30 min to ensure complete cell lysis, luminescence was measured using Envision (PerkinElmer, Waltham, MA, USA). The relative caspase 3/7 activity of lagunamide D treated cells were normalized to solvent control.

2.5. Mitochondrial membrane potential assay

The ability of lagunamide D to directly affect mitochondrial activity at early treatment time points was tested using MITO-ID^® Membrane Potential Cytotoxicity Kit (Enzo Life Sciences, ENZ-51019-KP002, Farmingdale, NY, USA) per manufacturer recommendations. HCT116 cells were seeded at 20,000/well in black-wall clear-bottom cell culture plates (Greiner Bio-One, Monroe, NC, USA) and left overnight to attach and acclimate. Cells were then treated with lagunamide D at 3-fold dilutions from 100 nM to 1 nM final concentration, or vehicle control (0.5% DMSO), and left to incubate at 37 °C for 1, 3, 12 or 24 h. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) provided with the kit was used as positive control at 10 μM final concentration with 30 minutes incubation at 37 °C as recommended. After incubation, Mito ID dye in assay buffer was added at 100 μL/well and the cells were incubated for additional 30 minutes with the dye at 37 °C. Orange fluorescence was measured using FlexStation3 monochromator plate reader (Molecular Devices) with 490 nm excitation and 590 nm emission filtering. Reduction in orange fluorescence indicated perturbation of the mitochondrial membrane potential status.

2.6. Cell cycle analysis

HCT116 cells were seeded at a density of 600,000 cells (in 2 mL DMEM with 10% FBS) per well in 6-well plates. After 24 h of incubation, the medium was removed and replaced with 1 mL fresh DMEM with 10% FBS. The cells were then treated with varying doses of lagunamide D or solvent control (DMSO). After 24 h of treatment, the medium in each well was collected into different tubes, and the cells were washed once with 500 μL Phosphate Buffered Saline (PBS) (collected into corresponding tubes). Cells were detached using 400 μL trypsin (Invitrogen) and 1400 μL DMEM with 10% FBS were added to neutralize trypsin. The cell suspensions were collected into corresponding tubes, centrifuged at 400g for 10 min at 4 °C. The supernatant was discarded, and the cell pellets were resuspended in 500 μL ice-cold PBS. The suspensions were centrifuged again at 400g for 10 min at 4 °C. The supernatant was discarded, and the cells were resuspended in 300 μL ice-cold PBS, and 700 μL ice-cold EtOH was slowly added to the cell suspension. Cells were incubated at −20 °C overnight, then centrifuged at 400g for 10 min at 4 °C. The EtOH/PBS was removed, and cells were resuspended in 300 μL PBS containing 1 mM EDTA and 100 μg/mL RNase A (Invitrogen). The cells were incubated at 37 °C for 30 min, while shaking at 800 rpm, followed by addition of 1 μL of propidium iodide (1 mg/mL, Invitrogen) to each tube. Fluorescence from propidium iodide−DNA complexes were quantified using FACScan (BD Biosciences LSRFortessa, San Jose, CA, USA) and data were analyzed using ModFit LT.

2.7. RNA extraction, cDNA synthesis and quantitative PCR (qPCR) analysis

HCT116 cells were seeded at a density of 80,000 cells per well in 6-well plates (2 mL cell suspension prepared in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin was dispensed into each well). After siRNA transfection or drug treatment with indicated time, total RNA was extracted using RNeasy mini kit (QIAGEN, Germantown MD, USA) according to the manufacturer’s instructions. RNA was quantified and qualified using a NanoDrop 2000 spectrophotometer. From 2 μg of total RNA, cDNA was synthesized by using SuperScript II Reverse Transcriptase and oligo(dT)_12–18 primer. qPCR was performed with a total reaction volume of 25 μL consisting of 12.5 μL of 2× TaqMan gene expression master mix, 1.25 μL of a 20× TaqMan gene expression assay probe, 0.33 μL of cDNA, and 10.92 μL of RNase-free sterile water. The reactions were dispensed into 96-well optical reaction plates and detected in a Real-Time PCR system using the thermocycler program: 2 min at 50 °C, 10 min at 95 °C, and 15 s at 95 °C (40 cycles) and 1 min at 60 °C. Each assay was performed in triplicate. PHB1 (Hs00855044_g1), PHB2 (Hs00200720_m1), and ATP1A1 (Hs00167556_m1) were used as target genes, while GAPDH (Hs02758991_g1) was used as endogenous control for normalization. Graphs and data analysis were performed using the GraphPad Prism 6.

2.8. RNA-sequencing and bioinformatic analysis

To assess transcriptional effects in an unbiased way, RNA-sequencing (RNA-seq) was performed. HCT116 cells were seeded at a density of 600,000 cells per well in 6-well plates. After 24 h of incubation, the cells were treated with 10 nM lagunamide D or vehicle (DMSO). After 3 h or 12 h of treatment, total RNA was extracted using RNeasy mini kit (QIAGEN) according to the manufacturer’s instructions. RNA was quantified and qualified using a NanoDrop 2000 spectrophotometer and submitted to the Interdisciplinary Center for Biotechnology Research (ICBR) at University of Florida (UF) for library generation and sequencing. In brief, RNA-seq library was constructed using NEBNext^® Ultra^™ Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) following manufacturer’s recommendations. Basically, 1000 ng of high-quality total RNA (RIN≥ 7) was used for mRNA isolation using NEBNext Ploy(A) mRNA Magnetic Isolation module (New England Biolabs, E7490). Then followed by RNA library construction with NEBNext Ultra II Directional Lib Prep (New England Biolabs, E7760) according to the manufacturer’s user guide. Briefly, RNA was fragmented in NEBNext First Strand Synthesis Buffer by heating at 94 °C for desired time. This step was followed by first strand cDNA synthesis using reverse transcriptase and oligo dT primers. Synthesis of double stranded cDNA was performed using the 2nd strand master mix provided in the kit, followed by end-repair and adaptor ligation. The library was enriched (each library has a unique barcode) by 11 cycles of amplification, and purified by Agencourt AMPure beads (Beckman Coulter, A63881, Indianapolis, IN, USA). Finally, eight individual libraries were pooled with equimolar and sequenced by Illumina HiSeq 3000 2×100 cycles run for total of 1 run (Illumina Inc., San Diego, CA, USA).

Short reads from Illumina sequencing were trimmed to remove low-quality bases using Trimmomatic[13] and analyzed with FastQC [14] to produce quality control reports. Reads were then mapped to the reference transcriptome using the STAR aligner[15] and gene expression was quantified using RSEM[16]. Differential gene expression was assessed by comparing the treated samples at the 3 h and 12 h timepoints against the DMSO control (2 replicates of each condition were analyzed). ANOVA was used to identify genes showing a significant difference in at least one of the tested conditions, while pairwise comparisons were performed using limma (BioConductor) to generate lists of significantly differentially expressed genes. Custom R scripts were used to generate volcano plots and the heatmap showing hierarchical clustering of differentially expressed genes.

2.9. Confocal microscopy of mitochondria in lagunamide D-treated HCT116 cells

HCT116 cells were seeded at 30,000/well in 8-chamber glass slides. Cells were left to attach for 24 h and then treated with lagunamide D at 10 nM or 3.2 nM. Cells were left to incubate for 1, 3, 6, 12 or 24 h. Ouabain treatment at 320 nM, 100 nM and 32 nM was also added as control. Cells were stained with 50 nM MitoTracker CMTMRos (Invitrogen, M7510) for 30 minutes and then fixed with 4% paraformaldehyde, followed by permeabilization in phosphate-buffered saline supplemented with 0.2% Triton X-100 and 1mg/mL bovine serum albumin, staining with Phalloidin AF488 (Invitrogen, A12379; 1:40 dilution in permeabilization buffer) and mounting with VectaShield containing DAPI nuclear stain (Vector Laboratories, H-1200, Newark, CA, USA). Slides were imaged on a Leica TSP5 confocal microscope at 40× with 5× smart zoom and 0.3 μm z-section step intervals to allow for detection of mitochondrial fragmentation.

2.10. RNA interference assay

HCT116 cells were seeded at a density of 4000 cells per well in 96-well plates (each well was dispensed 100 μL cell suspension prepared in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin). After 24 h of incubation, the medium was carefully aspirated and replaced with 50 μL of DMEM medium supplemented with 20% FBS. The transfection mixture was composed of indicated siRNAs (Invitrogen) and siLentFect transfection reagent (Bio-Rad Laboratories, Hercules, CA, USA) in a ratio of siLentFect/siRNA = 2 μL/1 μg in OptiMEM medium, and 50 μL of the mixture was dispensed to each well. Wells with negative control (cells + Silencer Negative Control No. 2 siRNA + solvent control) and medium only were also incorporated for determination of 100% viability and background level, respectively. After 48 h of transfection, the cells were exposed to varying concentrations of lagunamide D for 48 h and metabolic activity was measured using MTT assay.

2.11. Compound combination assays

In the combination study to investigate ATP1A1 as a potential target of lagunamide D, HCT116 cells (density as 4000 cells/well) and HeLa cells (density as 1200 cells/well)were pretreated with indicated doses of Na⁺/K⁺‑ATPase inhibitors ouabain or 3,4,5,6-tetrahydroxyxanthone for 1 h, followed by the treatment with indicated doses of lagunamide D. Cells were incubated for corresponding hours of treatment as noted in each figure before the addition of the MTT reagent (Promega).

A combination study to determine the effect of the proteasome inhibitor MG132 was carried out in HCT116 cells. Cells were seeded at 8000 cells/well in 96-well flat bottom plates and were pre-treated for 6 h with 125 nM of MG132 followed by 1.25 nM of lagunamide D. Lagunamide D only, proteasome inhibitor only, and 0.5% DMSO (vehicle) controls were included. Metabolic activity was measured at 48 h post lagunamide D treatment using MTT.

2.12. Genomic siRNA drug susceptibility screen (chemogenomics)

The siRNA library targeting 7,784 genes of the druggable human genome with quadruplicate coverage (four individual siRNAs for each gene) arrayed in 96-well format (Silencer^® Human Druggable Genome siRNA Library V3.1) was obtained from Ambion/Life Technologies (Grand Island, NY, USA). The siRNAs were spotted into 384-well screening sets (with no siRNA in column 23 and 24, reserved for positive and negative controls). Here two sets were used, one for lagunamide D treatment, the other for solvent control (DMSO). HCT116 cells (1000 cells per well, DMEM/5% FBS) were retro-transfected using siLentFect (Bio-Rad Laboratories) in OptiMEM (20 nM of siRNAs). ATP1A1 siRNA served as positive control and Silencer Negative Control No. 2 siRNA served as negative control. The plates were incubated at 37 °C and 5% CO₂ for 48 h. Lagunamide D (final concentration 4 nM) or solvent control (DMSO) containing DMEM was then dispensed into the corresponding plate set. After 48 h of incubation, cell viability was detected using ATPlite 1step according to the manufacturer’s instructions (PerkinElmer). Briefly, the plate and the reagent were equilibrated to room temperature for 15 min, and 50 μL of the assay reagent (2-fold diluted with H₂O) was dispensed to each well. After incubating in the dark for 10–15 min, the luminescence was recorded on SpectraMax M5. Hits from the siRNA library screen were considered as those where the viability ratio of the lagunamide D treatment to DMSO was <0.65 with p < 0.05 and high-confidence hits were selected if ≥ 2 hits were identified across the quadruplicate siRNA coverage, which was subjected to Ingenuity Pathway Analysis (IPA).

2.13. Proteasome inhibition assay

The effect of lagunamide D on the human proteasome in vitro was assessed using a 20S proteasome assay kit for drug discovery (Enzo Life Sciences, #BML-AK740) according to manufacturer instructions. Briefly, assay buffer was dispensed at 40 μL/well into white opaque half-area 96-well plate (PerkinElmer) and equilibrated to assay temperate (30 °C) for 10 minutes. The provided 20S proteasome was diluted to 20 μg/mL final concentration in assay buffer and added to the pre-warmed buffer at 10 uL/well. Epoxomycin was used as positive control at 500 nM final concentration as recommended. Lagunamide D was tested at 1.25 nM, 625 pM and 312 pM final concentrations. MG132 was tested at 125 nM, 62.5 nM and 31.2 nM final concentrations. The tested concentrations of lagunamide D and MG132 were selected to match the previously observed synergistic effect for the two drugs, and confirm that the observed synergy is not due to an additive proteasome-inhibition effect of lagunamide D. Compounds were incubated with the proteasome for 10 minutes at 30 °C, proteasome substrate was added and fluorescence was measured on FlexStation3 monochromator plate reader (Molecular Devices) with 355 nm excitation and 460 nm emission filtering every 1.5 minutes for 1 h.

2.14. Cellular thermal shift assay

The mitochondria suspension freshly isolated from HCT116 cells using the Mitochondria Isolation Kit for Cultured Cells (Invitrogen), following the manufacturer’s instructions, was pretreated with 100 nM lagunamide D or 0.5% DMSO for 1 h (37 °C, 750 rpm), which was then dispensed into 12 subfractions (using 0.2 mL PCR microtubes) and each aliquot was individually heated to corresponding temperature for 10 min. After cooling down to room temperature, the samples were lysed using a lysis buffer (PBS containing 0.1% NP-40 + 1% protease inhibitor cocktail) in addition to three freeze-thaw cycles in dry ice-ethanol bath. The samples were then centrifuged at 16,000g for 10 min at 4 °C to remove the insoluble fractions, and the supernatants were collected for immunoblot analysis. Protein was quantified using a BCA assay kit (Pierce, Rockford, IL). Equal volumes of samples were separated using SDS-PAGE (NuPAGE^™ 4–12% Bis-Tris polyacrylamide gels with NuPAGE^™ MES SDS running buffer, Invitrogen) and transferred onto polyvinylidene difluoride (PVDF) membranes (with NuPAGE^™ transfer buffer, Invitrogen). Each membrane was blocked with 5% BSA for 1 h at room temperature and incubated with indicated primary antibody (mouse anti-PHB1) overnight at 4 °C. After washing with TBST buffer for 3 times, the membranes were then incubated with secondary antibody (IRDye^® anti-mouse 800, LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature. After washing with TBST buffer for 5 times, images were taken by Odyssey infrared imaging system (LI-COR Biosciences) and the band intensities were quantified using Image Studio.

3. Results and Discussion

3.1. Antiproliferative effects induced by lagunamide D and differential susceptibility among cell sub-populations.

Lagunamide D was first evaluated in HCT116 human colorectal carcinoma cells at different time points (3, 12, 24 and 48 h) using the MTT assay (Figure 1B). Lagunamide D impaired the metabolic activity of HCT116 cells at single-digit nanomolar concentrations (IC₅₀ 5.1±0.8 nM after 48 h treatment) and in a time-dependent manner. Of note, since the MTT assay monitors the reduction of 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to MTT-formazan[17], which is partially correlated to mitochondrial succinate dehydrogenase, this assay is also applied as an indicator to assess mitochondrial function.[18] As indicated in Figure 1B, after only 3 h of treatment, metabolic activity was reduced by 50%, with apparent complete inhibition at 48 h. This finding suggested that lagunamide D exerts rapid action on perturbing the function of mitochondria in a similar manner to its analogue aurilide[9].

To explore the potential mitochondrial involvement in the observed effect by lagunamide D treatment, we employed parallel assays using trypan blue (Figure 1C) and mitochondrial membrane potential measurement (Figure 1D). Trypan blue measures cell viability independently of mitochondrial or metabolic effects. At 3 and 12 h time points, lagunamide D treatment showed minimal cytotoxicity by trypan blue in comparison to the effect measured by MTT at the same time. This suggests mitochondrial targeting at earlier time points (3 and 12 h) with possible cytotoxicity after 24 h where both MTT and trypan blue showed comparable effects (Figures 1B and 1C). The direct effect on mitochondria was further validated using a mitochondrial membrane potential assay, where lagunamide D treatment induced rapid membrane depolarization within 1 h of treatment at concentrations above IC₅₀ range (> 10 nM, Figure 1D). Another method we employed to test for early signs of intrinsic apoptosis was by measuring caspase 3/7 activity in lagunamide D treated cells at 3, 6 and 12 h post treatment. The time-dependent increase in caspase 3/7 activity, although slight, suggested the onset of apoptosis during the time frame (Figure 1E), indicating that the cell death detected by trypan blue at later time points possibly results from triggering caspase-independent mechanisms[19].

Closer inspection of the data at 3 h (Figure 1B), suggested partial or reduced efficacy, possibly indicating that a sub-population of cells responded more quickly to lagunamide D and that different cell sub-populations have a differential susceptibility. Based on this observation, the cell cycle stage dependent susceptibility to lagunamide D treatment was tested in cells specifically arrested at different cell cycle stages (Figures 2A–2C). We used metabolic activity (MTT reduction) as the readout for differential susceptibility, although at later time points it can be used as a proxy for cell viability (see Figures 1B and 1C). Cells arrested at G₀/G₁ phases were more sensitive towards lagunamide D, while cells synchronized at G₂/M phase had a delayed response. The differential efficacy of lagunamide D was evident at the 12 h time point, where the efficacy against cells at G₀/G₁ phases was 116%, relative to the entire cell population, while the efficacy against cells at G₂/M phase was 79%. Next, we performed FACS-based DNA content analysis to investigate the effect of lagunamide D on the cell cycle distribution (Figures 2B and 2C). It appeared that after 24 h the residual live cells with 32 nM lagunamide D treatment were largely arrested in G₂/M phase, increasing from 11% (vehicle control) to 36%, while the G₁ population was reduced from 57% (vehicle control) to 28%. Taken together, the data suggests preferential cytotoxic effects on the G₁ population at higher concentrations. This observation further supports the direct effect of lagunamide D on mitochondrial function perturbation, as mitochondria are well known to play a major role in cell cycle progression and proliferation, especially in the growth phases[20–22].

Figure 2:

Cell cycle analysis and transcriptome profiling for lagunamide D treated HCT116 cells. (A) Left: Antiproliferative effect of lagunamide D on non-arrested HCT116 cells at different time points, measured by MTT assay. Middle: Cells were arrested at G₀/G₁ stage by starving for 24 h with low-serum medium (DMEM, 0.5% FBS, and 1% penicillin/streptomycin) before lagunamide D treatment. Antiproliferative effect was measured using MTT assay. Right: Cells were arrested at G₂/M phases by treating with 650 nM nocodazole for 24 h and harvested by mechanical shaking. Cell viability was measured after treatment with lagunamide D for 12 or 48 h, respectively, using MTT assay. Data are presented as mean ± SD (n = 3), relative to 0.5% DMSO treatment. (B) Cell cycle distribution in HCT116 cells after treatment with different concentrations of lagunamide D for 24 h. DNA content was determined by flow cytometric analysis (C) Quantification of cell cycle distribution data. Transcriptome profiling (D-F) of HCT116 cells treated with 10 nM lagunamide D and solvent control. (D) Mean-difference plot for 3 h. Red dot indicated up-regulation and green indicated down-regulation with adjusted p-value < 0.05. 2887 genes were identified beyond the 1.5-fold change cutoff (dotted line). (E) Mean-difference plot for 12 h. Red dot indicated up-regulation and green indicated down-regulation with adjusted p-value < 0.05. 58 genes were identified beyond the 1.5-fold change cutoff (dotted line). (F) Heatmap for transcript changes in HCT116 cells after 3 and 12 h treatment with 10 nM lagunamide D.

3.2. RNA-seq analysis confirms possible mitochondrial involvement in lagunamide D activity.

To obtain an unbiased global view of how lagunamide D functions in cells, transcriptome profiling using RNA-sequencing analysis was performed (Figures 2D–2F). Lagunamide D rapidly perturbed gene expression after 3 h of treatment, where 2887 differentially expressed genes were identified with high confidence (Figures 2D–2F). This effect of lagunamide D was largely transient as residual live cells after 12 h of treatment showed only few gene expression changes (58 hits, Figure 2E).

Ingenuity Pathway Analysis (QIAGEN) was performed on the 3 h treatment with 1.5-fold change cutoff increase or decrease in gene expression, and p-value cutoff at 0.05. With these selected cutoffs 275 analysis ready up- or down-regulated molecules were detected. Those were analyzed for “Top Diseases and Bio Functions” and “Top Upstream Regulators and Causal Networks”. The top disease detected by the analysis was Cancer (p-value range 0.0489 – 0.00000325), with 250 molecules of which 246 were “carcinoma” associated, while the top molecular and cellular function was “Cell cycle” (p-value range 0.048 – 0.000149). Interestingly, the top 2 causal networks were related to mitochondrial functions, involving OPA1 (p-value 0.0013) mitochondrial inner membrane protein (a regulator of mitochondrial stability and energy output) and LOC102724788/PRODH (p-value 0.00265) proline dehydrogenase 1 (mitochondrial matrix and inner membrane protein), both of which play a role in mitochondrial processes and intrinsic apoptosis (OPA1; PRODH).[23,24] From a search for the top analysis-ready molecules the most upregulated was CORO7/CORO7-PAM16 (coronin 7-PAM16; 5-fold change) where coronin 7 is an F-actin regulator directing anterograde Golgi to endosome transport, while PAM16 is a mitochondrial import inner membrane translocase subunit important for protein translocation into mitochondria. The most downregulated molecule, C8orf44-SGK3 (4-fold change), is a serine/threonine-protein kinase which is involved in the regulation of a wide variety of ion channels and membrane transporters, cell growth, proliferation, survival/apoptosis, and migration.[25,26]

Additionally, 18 of the top analysis molecules were associated with apoptosis and 8 with cell cycle (with 4 molecules overlapping). Among those DDIT3 (DNA damage inducible transcript 3, a.k.a. C/EBP homologous protein (CHOP)) is known to be related to apoptosis of carcinoma cells[27] and IRF5 (interferon regulatory factor 5) is known for involvement specifically in colon carcinoma cell apoptosis[27]. Several molecules related to cell cycle progression/arrest were also noted in this set: KRT13 (keratin 13) and SSTR5 (somatostatin receptor 5) have been associated with G₁-S transition and arrest,[28,29] while FADD (Fas associated via death domain) has been linked to arrest in G₂/M phase of the cell cycle[30]. APOE (apolipoprotein E) is known to inhibit G₁ to S phase progression by repression of the cyclin A promoter[31]; however, in lagunamide D treated cells APOE was significantly downregulated, suggesting opposite effect. Mitochondria and their dynamics are well known for their importance to the cell cycle progression[21,32] and mitochondrial replication and membrane potential have been shown to increase from G₁ to S phase of the cell cycle[20] which may explain why cells arrested in G₁ phase are more sensitive to perturbation of mitochondrial function. Persistent defects in mitochondrial fusion/fission dynamics have also been shown to induce arrest in G₂/M phase[33], and lead to cell death via a caspase 8-dependent mechanism.

3.3. Confocal microscopy confirms mitochondrial rearrangement without fragmentation.

The effect of lagunamide D on mitochondria was directly monitored using confocal microscopy. Mitochondria were pre-stained with MitoTracker^™ CMTMRos, a probe that enables mitochondria visualization and the assessment of mitochondrial dynamics. Mitochondrial fragmentation was not observed at any time point or tested concentrations of lagunamide D. Lagunamide D at 10 nM showed more cells with mitochondrial network redistribution around the nucleus compared to DMSO control, which was visible after 12 h of treatment, whereas 3.2 nM lagunamide D had no observable effect on mitochondria at any of the tested time points (Figures 3A). In the majority of the HCT116 cells with any treatment mitochondrial network was oriented along the z-plane, and regions that appeared “dotted” or “fragmented” on maximum image projections still showed long tube-like organization along the z-plane when viewed in orthogonal section mode (Figure 3B). Same re-distribution around the nucleus as previously reported[34] was observed with Ouabain, which is a known Na⁺/K⁺-ATPase inhibitor[35], at 320 nM dose tested, but along the z-plane level mitochondria were also longitudinally oriented as observed with lagunamide D (Figure 3B). This result was in agreement with our previous finding about the fast-acting manner of lagunamide D indicative of disturbing mitochondrial function.[3] Moreover, given the pivotal role of PHB1 in the mechanism of action of aurilide, in regulating mitochondrial fusion and maintaining mitochondrial integrity,[36–39] we further explored PHB1 target against lagunamide D.

Figure 3:

Confocal microscopy of mitochondrial distribution in lagunamide D treated HCT116 cells. (A) HCT116 treated with Lagunamide D at 10 nM or 3.2 nM for 6, 12 or 24 h (maximum image projections). (B) Orthogonal sectioning of HCT116 treated with 10 nM or 3.2 nM Lagunamide D for 24 h, compared to vehicle-treated cells (DMSO). Ouabain at 320 nM for 12 h was used as a control. Cells were stained with Alexa Fluor 488-Phalloidin (green), MitoTreacker CMTMRos (red) and the nuclear stain DAPI (blue). Scale bars represent 15 μm. Aggregation of the mitochondria in cells is shown with white arrowhead. Longitudinal orientation of mitochondrial tubes along the z-stacks are shown with yellow arrowheads on the orthogonal sections in B.

3.4. Effects of lagunamide D on specific mitochondrial target candidates.

Based on the demonstrated effects on mitochondria and the structural similarity to aurilide, it was suspected that lagunamide D might share the same mitochondrial molecular target with aurilide. To explore this hypothesis, we aimed to use cellular thermal shift assay (CETSA) to directly monitor target engagement in a cellular context by taking advantage of the shift of protein thermal stability induced by ligand binding.[40–42] However, as shown in Figure 4A, no significant shifts of the protein aggregation curves towards stabilization could be detected between the lagunamide D treatment group and vehicle control. A small shift towards destabilization was indeed observed, which may or may not be relevant. While CETSA has been proven to be a powerful method to monitor direct target engagement for various molecules, it has its intrinsic limitations which might lead to the occurrence of false negative results. It is especially challenging to apply this method for membrane protein targets, as their structures are often stabilized by the lipid bilayer and therefore more likely to give weak or no ligand-induced response in thermal shift assays.[41,42] In addition to the fact that PHB1 is anchored at mitochondria inner membrane, it is also known to assemble with its homologue PHB2 to form large ring-shaped heterodimer complexes.[43–45] Under this circumstance, the obtained results could not be viewed as a conclusive evidence about the direct interaction between lagunamide D and PHB1. Since PHB1 silencing was reported to be able to sensitize HeLa cells to aurilide,[9] a siRNA knockdown assay was performed to investigate whether lagunamide D could cause the same effect in CRC cells. The IC₅₀ of lagunamide D against HCT116 was not appreciably changed upon knockdown of PHB1 or in combination with PHB2 silencing; however, PHB2 knockdown in HCT116 cells reduced mitochondrial activity and cell viability (Figures 4B and 4C). In a similar study using HeLa cells, PHB1 knockdown had also failed to potentiate the antiproliferative activity of aurilide B,[46] a close analogue of aurilide (only differing in one amino acid, N-Me-Ile versus N-Me-Leu). This discrepancy further raised a concern about the similarity among aurilides and lagunamides and their functional equivalency with respect to the relevant molecular target(s) and MOA.

Figure 4.

CETSA and RNAi-mediated PHB1, PHB2, and ATP1A1 knockdown on the susceptibility of cells towards lagunamide D. (A) CETSA. The suspension of freshly isolated mitochondria were pre-treated with 100 nM lagunamide D or DMSO for 1 h at 37 °C and then heated to corresponding temperature for 10 min. The PHB1 level was quantified using immunoblot analysis. For the CETSA curves, the band intensity of each sample was correlated to the intensity of the lowest tested temperature (46 °C) within each set. No significant shift of the CETSA curve was observed between lagunamide D treated group and DMSO control. Data are presented as mean ± SD from two independent experiments. (B and C) Targeted studies to probe PHB1 as the molecular target of lagunamide D using siRNA knockdown. MTT assay was used as a proxy for cell viability at the 48 h time point. (B) HCT116 cells were transfected with indicated siRNA(s) for 48 h and then treated with varying doses of lagunamide D for another 48 h. Cell viability was measured using MTT assay. Silencing PHB1 or PHB2 alone or in combination did not significantly affect the IC₅₀ of lagunamide D but suppressed the dynamic range and efficacy due to inherent effects of the siRNAs. Data are presented as mean ± SD (n = 3), relative to the negative control (20 nM or 40 nM NG siRNA (Silencer Negative Control No. 2 siRNA) + 0.5% DMSO treatment). (C) Knockdown efficiency in HCT116 cells (48 h), using GAPDH as the endogenous control. Data are presented as mean ± SD (n = 3), **** p-value < 0.0001, compared to NG siRNA-transfected cells (Student’s t test). (D) HCT116 cells were transfected with 20 nM ATP1A1 siRNA or 20 nM negative control (NG) siRNA (Silencer Negative Control No. 2 siRNA) for 48 h and then treated with varying doses of lagunamide D for another 48 h. Cell viability was measured using MTT assay. Data are presented as mean ± SD (n = 3), relative to the negative control (20 nM negative control siRNA + 0.5% DMSO treatment). Silencing ATP1A1 could efficiently sensitize HCT116 cancer cell line to lagunamide D. The IC₅₀ of lagunamide D was shifted from 5.4 ± 1.3 nM to 2.2 ± 0.6 nM (2.5-fold). (E) Knockdown efficiency of 20 nM ATP1A1 siRNA in HCT116 cells. Total RNA was extracted after the cells were transfected with indicated siRNA for 48 h. After cDNA synthesis, qPCR was carried out to measure the transcript level of ATP1A1 while using GAPDH as the endogenous control. Data are presented as mean ± SD (n = 3), **** p-value < 0.0001, compared to NG siRNA-transfected cells (Student’s t test).

It was previously found that siRNA-mediated knockdown of ATP1A1, encoding the α-subunit of Na⁺/K⁺ ATPase complex, could potentiate the antiproliferative activity of aurilide B against HeLa S3 cells,[46] which prompted us to investigate effects of ATP1A1 knockdown on lagunamide D activity. The sensitizing effect was also detected using HCT116 cells (Figures 4D and 4E), where a 2.5-fold increased potency was observed. Next, to investigate whether inhibition of Na⁺/K⁺ ATPase using a small molecule inhibitor could mimic the effect of siRNA mediated ATP1A1 knockdown on sensitizing cells to lagunamide D, a combination study was performed with lagunamide D and ouabain, a well-characterized Na⁺/K⁺ ATPase inhibitor, as the drug pair. To potentially validate our findings and assess generalizability, we used HeLa human cervical carcinoma cells, which possessed with different genetic background and was previously used in aurilide studies.[46] Dose-response curve was first plotted for ouabain to determine its IC₅₀ value in both HCT116 and HeLa cells (Figures 5A and 5B), which would be a critical parameter for selecting the range of concentrations used in the combination study. Synergy was assessed by treating the cells with a two-dimensional array of concentrations of the two compounds in a checkerboard assay matrix and data quantified by the deviation from Bliss independence (Bliss score), which is a method widely applied for drug combination studies.[47–50] As shown in Figures 5C and 5E (left panel), a synergistic effect could be consistently observed between the compound pair in both tested cell lines. Notably, the synergistic effect occurred at concentrations (low-nanomolar range, ≤50 nM) where the pump activity of Na⁺/K⁺ ATPase was expected to be unaffected, as the IC₅₀ of Na⁺/K⁺ ATPase activity was reported to be at micromolar range (≥ 1.0 μM).[51–55] Moreover, it has been shown that the growth-regulatory effects of ouabain are highly dependent on cell type, and cancer cells appear to be much more susceptible to this compound than nontumorigenic counterparts.[51,55–59] Under this circumstance, the effective concentration of ouabain (6.25, 12.5 nM) that produces significant synergy was possibly within a relatively safe range. In addition, it should be noted that the cell viability was still maintained (77% in HCT116 cells and 95% in HeLa cells) when treating with 2.5 nM lagunamide D alone, the concentration that produced an optimal synergistic effect (Figures 5D and 5F). This combination may indicate promise for lagunamide D to be administrated at a relatively nontoxic dose while still maintaining efficacy.

Figure 5.

Combination studies of lagunamide D and Na⁺/K⁺-ATPase inhibitors. (A and B) Antiproliferative effects of the Na⁺/K⁺-ATPase inhibitor ouabain and 3,4,5,6-tetrahydroxyxanthone against HCT116 cells (A) and HeLa cells (B). The IC₅₀ value against HCT116 cells for ouabain was 18.9 ± 2.6 nM and for 3,4,5,6-tetrahydroxyxanthone was 28.2 ± 5.2 μM. The IC₅₀ value against HeLa cells for ouabain was 13.6 ± 1.3 nM,and for 3,4,5,6-tetrahydroxyxanthone was 31.8 ± 8.1 μM. Data are presented as mean ± SD (n = 3), relative to 0.5% DMSO treatment. (C and E) Δ Bliss independence calculations in HCT116 cells (C) and HeLa cells (E) for the combination of ouabain + lagunamide D (left) and 3,4,5,6-tetrahydroxyxanthone + lagunamide D (right). Cells were pre-treated with ouabain or 3,4,5,6tetrahydroxyxanthone at the indicated concentrations for 1 h, followed by the treatment of lagunamide D at the indicated concentrations. Cell viability was measured using MTT assay after a total of 48 h of drug exposure. “Δ Bliss independence” is the difference between observed growth inhibition and Bliss expectation. Values greater than zero represent a synergistic response. Bliss expectation equals is C = (A + B) – (A × B), where A and B are the growth inhibition fractions of two compounds at a given dose and expressed relative to the negative control (0.5% DMSO). * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, relative to the expected inhibitory effect of the corresponding two compounds (Student’s t test, n = 3). (D and F) The antiproliferative effects of lagunamide D alone and ouabain alone versus the drug combination at indicated dose tested in the combination study. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001, (ANOVA Tukey’s test, n = 3 for drug treatments, n=12 for solvent control).

Na⁺/K⁺ ATPase is involved in multiple cellular processes beyond pumping ions and modulating membrane potential but also plays a role as intracellular signaling transducer to initiate kinase signaling cascades.[53–55,60] To explore the underlying mechanism of synergy, a second Na⁺/K⁺ ATPase inhibitor, 3,4,5,6-tetrahydroxyxanthone (Figures 5C and 5E, right panel)), was included in the combination studies. It was chosen due to its distinct characteristics in inhibiting the Na⁺/K⁺ ATPase, as it could abrogate the pumping function of Na⁺/K⁺ ATPase in a similar potency as ouabain without activating its role as signal transducer.[52] As expected, no synergistic effect was detected between this compound and lagunamide D in both tested cell lines (Figures 5C and 5E, right panel), which further verified the previously observed synergy between ouabain and lagunamide D was not due to the disturbed pump function or the subsequent disrupted ion homoeostasis. As the function of Na⁺/K⁺ ATPase is highly dependent on ATP, it was plausible that lagunamide D might affect cellular ATP level (due to its effects on mitochondria function), and therefore produce the synergistic effect. However, at the effective concentration for significant synergy (2.5 nM of lagunamide D), the ATP level was unaffected after 48 h of treatment. Therefore, cellular ATP level was less likely to be a critical factor in yielding the synergistic effect. Although the mechanism behind the synergy remained unclear, ruling out two potential possibilities (pump function/ionic environment and ATP level) made the signaling pathways modulated via ATP1A1 to be new candidates for further analysis. Synergy might be a result of overlapping or complementary signaling events. Moreover, the similar behavior of lagunamide D and aurilide B in the siRNA knockdown assay and drug combination assay indicated these two compounds putatively share a similar MOA.

3.5. Genome-wide RNAi screening (chemogenomics).

We extended our chemical-genetic interaction analysis from ATP1A1 to a genome-wide level by performing a genomic RNAi screen to provide more unbiased information about the complex MOA for lagunamide D and to decipher cancer cell vulnerabilities that could be exploited for other types of combination therapy. The utilized arrayed siRNA library targeted the human druggable genome (7784 genes) with quadruplicate coverage (four siRNAs per gene). The overview of the procedures is depicted in Figure 6A, which involved high-throughput retro-transfection, compound/control treatment, cell viability measurement and data analysis. In addition, ATP1A1 siRNA and Silencer Negative Control No. 2 siRNA (Invitrogen) were spotted into each plate to serve as positive and negative controls, respectively. As shown in Figure 6B, the positive control (color-coded in yellow) possesses a clear separation with the negative control (color-coded in green), indicating the appropriate choice of the screening conditions. Hits were determined with the criteria that the viability ratio of the lagunamide D treatment / DMSO was ≤ 0.65, while high confidence hits were the ones identified with ≥ 2 siRNA hits targeting the same transcript. In total 52 high-confidence hits (Figure 6B, highlighted in red;) were identified with the potential to sensitize cells against lagunamide D, among which, 3 genes: VDAC3, RPN2, and PIAS2, were identified with 3 siRNA hits (Figure 6C). The identification of VDAC3 (voltage-dependent anionselective channel protein 3), a mitochondrial protein, as a putative hit is in agreement with others’ reports and our finding that aurilides and lagunamide D might exert effects via disturbing mitochondrial function. Of note, VDAC2 was previously shown to interact with PHB2.[61] In addition, 5 dual hits were ubiquitin specific peptidases (Figure 6C) and the triple hit PIAS2 (protein inhibitor of activated STAT2) functions as a E3 SUMO (small ubiquitin-like modifier)-protein ligase, suggesting that lagunamide D might also exert its effects via affecting proteostasis or can be modulated by affecting proteasome function. We then tested this hypothesis through biochemical and cellular assays. While lagunamide D did not directly inhibit 20S proteasome activity in an in vitro assay (Figure 6D), pretreatment of HCT116 cells with proteasome inhibitor MG132 sensitized cells to lagunamide D, mimicking the RNAi screen. Specifically, cells were treated with MG132 for 6 hours (at IC₂₅) before adding low concentration of lagunamide D (at ~IC₅), producing a cooperative effect (Figure 6E). Specifically, the viability was reduced to 56% under those conditions.

Figure 6.

Genome-wide RNAi screening. (A) Overview of the high-throughput genome-wide RNAi screening procedure. (B) Hits were determined with the criteria that the viability ratio of the lagunamide D treatment / DMSO was ≤ 0.65, while high confidence hits (shown in red) were the ones identified with ≥ 2 siRNA hits targeting the same gene. Positive control, ATP1A1 siRNA, was color-coded in yellow. Negative control, Silencer Negative Control No. 2 siRNA, was color-coded in green. (C) Gene list of the hits with triple siRNA targeting the same transcript (top) and gene list of the hits involved in ubiquitin/proteasome system (bottom). (D) Lagunamide D (LagD) effect on human 20S proteasome in vitro. Lagunamide D (2-fold dilution) and MG132 (2-fold dilution) tested at concentrations used for the combination studies and lower to verify lack of additive effect observed in the combination assay. Epoxomycin (500 nM) was used as the proteasome inhibitor control. Data displayed in arbitrary fluorescence units (AFU). (E) Combination study of lagunamide D (1.25 nM) and proteasome inhibitor (MG132, 125 nM). HCT116 cells were pre-treated for 6 h with the proteasome inhibitor and then treated with lagunamide D for 48 h. Cell viability was measured using MTT assay.

In conclusion, the global and targeted MOA studies enabled in-depth understanding of the cellular effects of lagunamide D. Lagunamide D exerts cytotoxicity via affecting mitochondrial functions likely in the same manner as aurilides. However, unlike reported for aurilides, lagunamide D did not cause mitochondrial fragmentation, but a mitochondrial network rearrangement instead, which may be less severe and tunable. A chemogenomic approach identified cancer cell vulnerabilities and suggested pathways that may be targeted concurrently to achieve cooperative effects with lagunamide D. The identification of synergistic drug combinations, exemplified by proteasome inhibitors, increased the potential for lagunamide D to possess therapeutic use with a reduced dose to alleviate potential toxic side effects and improve the safety profile of this compound.

Abbreviations

AFU

arbitrary fluorescence units

ANOVA

analysis of variance

APOE

apolipoprotein E

ATP1A1

ATPase Na⁺/K⁺ transporting subunit alpha 1

BCA

bicinchoninic acid

C8orf44

chromosome 8 putative open reading frame 44

CCCP

carbonyl cyanide m-chlorophenylhydrazone

CETSA

cellular thermal shift assay

CHOP

C/EBP homologous protein

CMTMRos

chloromethyltetramethylrosamine

CORO7

coronin 7

CRC

colorectal cancer

DDIT3

DNA damage inducible transcript 3

DMEM

Dulbecco’s modified Eagle medium

EtOH

ethanol

FACS

fluorescence-activated cell sorting

FADD

Fas associated via death domain

FBS

fetal bovine serum

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

IC₅

5% inhibitory concentration

IC₂₅

25% inhibitory concentration

IC₅₀

half-maximal inhibitory concentration

IPA

ingenuity pathway analysis

IRF5

interferon regulatory factor 5

KRT13

keratin 13

LagD

lagunamide D

MES

2-(N-morpholino)ethanesulfonic acid

MG132

carbobenzoxy-L-leucyl-L-leucyl-L-leucinal

MOA

mechanism of action

MTT

3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide

OPA1

optic atrophy 1 mitochondrial dynamin like GTPase

PAM16

presequence translocase associated motor 16

PHB1

prohibitin 1

PHB2

prohibitin 2

PIAS2

protein inhibitor of activated STAT 2

PRODH

proline dehydrogenase 1

PVDF

polyvinylidene difluoride

qPCR

quantitative polymerase chain reaction

RNAi

RNA interference

RNA-seq

RNA-sequencing

RPN2

ribophorin 2

SD

standard deviation

SDS-PAGE

sodium dodecyl-sulfate polyacrylamide gel electrophoresis

SGK3

serum/glucocorticoid regulated kinase family member 3

siRNA

small interfering RNA

SSTR5

somatostatin receptor 5

SUMO

small ubiquitin-like modifier

TBST

tris-buffered saline with 0.1% Tween 20 detergent

VDAC3

voltage dependent anion channel 3