Abstract

   Pleurocidin-family cationic antimicrobial peptide NRC-03 exhibits
   potent and selective cytotoxicity towards cancer cells. However, the
   anticancer effect of NRC-03 in oral squamous cell carcinoma (OSCC) and
   the molecular mechanism of NRC-03 induced cancer cell death is still
   unclear. This study focused to investigate mitochondrial oxidative
   stress-mediated altered mitochondrial function involved in
   NRC-03-induced apoptosis of OSCC cells. NRC-03 entered the OSCC cells
   more easily than that of normal cells and bound to mitochondria as well
   as the nucleus, causing cell membrane blebbing, mitochondria swelling,
   and DNA fragmentation. NRC-03 induced high oxygen consumption, reactive
   oxygen species (ROS) release, mitochondrial dysfunction, and apoptosis
   in OSCC cells. Non-specific antioxidant N-acetyl-l-cysteine (NAC), or
   mitochondria-specific antioxidant mitoquinone (MitoQ) alleviated
   NRC-03-induced apoptosis and mitochondrial dysfunction indicated that
   NRC-03 exerts a cytotoxic effect in cancer cells via inducing cellular
   and mitochondrial oxidative stress. Moreover, the expression of
   cyclophilin D (CypD), the key component of mitochondrial permeability
   transition pore (mPTP), was upregulated in NRC-03-treated cancer cells.
   Blockade of CypD by siRNA-mediated depletion or pharmacological
   inhibitor cyclosporine A (CsA) significantly suppressed NRC-03-induced
   mitochondrial oxidative stress, mitochondrial dysfunction, and
   apoptosis. NRC-03 also activated MAPK/ERK and NF-κB pathways.
   Importantly, intratumoral administration of NRC-03 inhibited the growth
   of CAL-27 cells-derived tumors on xenografted animal models. Taken
   together, our study indicates that NRC-03 induces apoptosis in OSCC
   cells via the CypD-mPTP axis mediated mitochondrial oxidative stress.

   Keywords: NRC-03, Oral squamous cell carcinoma, Cyclophilin D, Cell
   apoptosis, Oxidative stress

Graphical abstract

   [41]Image 1
   [42]Open in a new tab

Highlights

     * •
       NRC-03 induces OSCC cell apoptosis and inhibits OSCC ectopic tumor
       growth in mice.
     * •
       NRC-03 binds to the plasma membrane via electrostatic interaction
       and enters into cells.
     * •
       NRC-03 binds to the mitochondrial membrane, enters the nucleus,
       induces mtROS.
     * •
       NRC-03 upregulates CypD expression that triggers mPTP opening and
       caspase cascade.
     * •
       NRC-03 causes mitochondrial dysfunction and DNA fragmentation to
       induce apoptosis.

1. Introduction

   Oral squamous cell carcinoma (OSCC) is the most common oral malignancy
   and represents around 90% of all cancers in the oral cavity [[43]1],
   causing approximately 300,000 cases and 145,000 deaths worldwide
   annually [[44]2,[45]3]. OSCC in the initial stages shows an
   asymptomatic erytholeukoplastic lesion mainly on the tongue, mouth
   floor, buccal mucosa, gingiva, and lips [[46]4]. In advanced stages of
   OSCC, the lesions may develop into ulcers and lumps with irregular and
   poorly defined margins, causing a series of severe symptoms, such as
   severe pain, bleeding, problems in breathing, and difficulty in speech
   [[47][5], [48][6], [49][7]]. Because early carcinomas are asymptomatic,
   most OSCC cases are usually diagnosed in advanced stages [[50]8].
   Moreover, OSCC exhibits high aggression, rapid progression, and early
   relapse, thus causing high mortality with a five-year survival rate of
   less than 50% [[51]9]. Chemotherapy is a critical treatment option in a
   multimodality therapeutic approach to treat locally advanced tumors,
   nonresectable tumors, metastatic tumors, and palliative chemotherapy
   [[52][10], [53][11], [54][12]]. Cisplatin, carboplatin, 5-fluorouracil,
   paclitaxel, and docetaxel are the commonly used first-line
   chemotherapeutic drugs to treat OSCC with a mechanism of damaging cell
   replication machinery of rapidly dividing cells [[55]6,[56]13].
   However, apart from cancer cells, these drugs also damage the healthy
   cells with a high division rate from bone marrow, hair follicles, and
   gastrointestinal tract, which leads to a series of severe side effects,
   such as myelosuppression, alopecia, rashes, and vomiting
   [[57]14,[58]15]. To approach this problem, continuous efforts have been
   taken to develop cancer-targeting chemotherapeutic drugs [[59]16].

   NRC-03, a 26-residue pleurocidin-like cationic antimicrobial peptides
   (CAPs), is derived from skin mucous secretions of winter flounder, and
   has promising cancer-targeting potential [[60]17]. The cationic
   property allows NRC-03 to specifically target negatively charged cancer
   cells, thereby causing membrane disturbance and cell death. Recent
   studies have shown that NRC-03 selectively kills human breast cancer,
   multiple myeloma, and leukemia cells, but is less toxic to normal
   cells, such as human umbilical vein endothelial cells, dermal
   fibroblasts, and erythrocytes [[61][18], [62][19], [63][20], [64][21]].
   However, the anticancer potential of NRC-03 towards OSCC has not been
   investigated yet. Furthermore, the mode and molecular mechanisms of
   NRC-03-induced cancer cell death remain largely unknown. One major
   subcellular target of CAPs is mitochondria which play an important role
   in tumor initiation and progression through ATP production, catabolic
   and anabolic metabolism, generation of ROS, and apoptosis [[65]22].
   Certain CAPs can interact with mitochondria directly or indirectly to
   trigger ROS generation, causing mitochondrial dysfunction and cancer
   cell death [[66][23], [67][24], [68][25], [69][26]]. In breast cancer
   cells, NRC-03 has been shown to co-localize with mitochondria, decrease
   mitochondrial membrane potential and cause the release of cytochrome c
   [[70]18]. However, the underlying molecular mechanisms remain unclear.
   mPTP is a reversible mitochondrial channel, which plays an important
   physiological role in maintaining mitochondrial homeostasis by the
   timely discharge of ROS and Ca^2+ from mitochondria [[71]27]. In
   pathological conditions, such as oxidative stress, continuous mPTP
   openings cause a burst release of ROS, resulting in mitochondrial
   dysfunction [[72]28,[73]29], which forms a vicious circle leading to
   cell death [[74]30]. CypD is a critical regulator for mPTP opening
   [[75]31]. CypD-dependent mPTP opening has been shown to play a key role
   in ROS-induced mitochondrial dysfunction and cell death
   [[76]31,[77]32]. However, the role of CypD-dependent mitochondrial
   dysfunction in NRC-03-induced cancer cell death is still unclear.

   In this study, we aimed to investigate the anti-OSCC efficacy of NRC-03
   and its underlying mechanisms. We first assessed the cytotoxicity,
   apoptosis, and DNA damage potential of NRC-03 in two OSCC cell lines
   (CAL-27 and SCC-9) and normal human oral keratinocytes (HOK). We
   further investigated the OSCC growth inhibition potential of NRC-03 in
   nude mice ectopic tumor model. Thereafter, we monitored the interaction
   between NRC-03 and OSCC cells and evaluated the role of mitochondrial
   dysfunctions in NRC-03-induced apoptosis of OSCC cells. RNA sequencing
   was adopted to sort out the involved signaling pathways. Finally, we
   proved the critical role of CypD-mPTP openings in the anti-OSCC effect
   of NRC-03.

2. Materials and methods

2.1. Peptide synthesis

   NRC-03 (GRRKRKWLRRIGKGVKIIGGAALDHL-NH[2]) and tetramethylrhodamine
   (TRITC)-labeled NRC-03 were synthesized by Top-peptide Co., Ltd.
   (Shanghai, China) via Fmoc solid-phase peptide synthesis. The purity of
   the peptide was over 95%. Molecular weight (MW): 2953.4, net charge:
   +9.5, and isoelectric point (pI):12.67 were the basic biochemical
   properties of NRC-03 ^19. Lyophilized peptides were reconstituted in
   serum-free Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12
   (DMEM/F-12). All experiments were conducted in a medium containing 2.5%
   FBS to limit peptide degradation by serum proteases.

2.2. Reagents

   Cell culture medium and supplements were purchased from GIBCO BRL
   (Gaithersburg, MD, USA). Anti-β-actin antibody (#4970) and anti-rabbit
   secondary antibody (#7074) were obtained from Cell Signaling Technology
   (MA, USA). Anti-CypD antibody (ab110324) and Hydrogen peroxide assay
   kit - (Fluorometric-Near Infrared) (ab138886) were from Abcam (MA,
   USA). Anti-mouse secondary antibody (sc-516102) was purchased from
   Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). MitoSOX Red
   Mitochondrial Superoxide Indicator, MitoTracker Green FM, and
   LysoTracker Green DND-26 were from Yeasen biotechnologies Co., Ltd.
   (Shanghai, China). Terminal deoxynucleotidyl transferase dUTP nick-end
   labeling (TUNEL) kit, Caspase-3 activity assay kit, Caspase-8 activity
   assay kit, Bradford protein assay kit, Adenosine triphosphate (ATP)
   assay kit, Hoechst 33342 staining kit, NAC, phenylmethanesulfonyl
   fluoride (PMSF), and RIPA buffer were from Beyotime Institute of
   Biotechnology (Shanghai, China). Z-VAD-FMK and cyclosporine A (CsA)
   were purchased from MedChemExpress (Princeton, NJ, United States).
   Mitoquinone (MitoQ) was from Cayman Chemical Company (Ann Arbor, MI,
   USA). Oxygen consumption rate (OCR) assay kit,
   2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), annexin
   V-FITC/propidium iodide (Annexin V-PI) staining kit, mitochondrial
   membrane potential assay kit with JC-1, and BCA protein assay kit were
   purchased from BestBio biotechnologies Co., Ltd. (Shanghai, China).
   Lipofectamine 3000 transfection reagents were from Invitrogen
   (Carlsbad, CA, USA).

2.3. Cell lines and cell culture

   OSCC cell lines CAL-27 and SCC-9 were purchased from ATCC and cultured
   in DMEM/F-12. HOK were obtained from ScienCell and cultured in Oral
   Keratinocyte Medium. The medium was supplemented with 10% fetal bovine
   serum (FBS) and 1% penicillin-streptomycin in 5% CO[2] at 37 °C in a
   humidified incubator.

2.4. Cell treatment

   Test compounds were prepared as stock solutions and diluted to the
   desired final concentrations immediately before use. The final
   concentrations of the compounds were as follows: NRC-03 (45 μg/ml),
   Z-VAD-FMK (50 μM), NAC (5 mM), MitoQ (1 μM), and CsA (2 μM). Cells were
   treated with or without NRC-03 and the indicated test compounds
   according to the experiment protocol.

2.5. Cell viability assay

   In vitro cytotoxicity was determined by the Cell Counting Kit-8
   (Dojindo Corp.) assay. Cells were plated in 96-well plates
   (1 × 10^4 cells/well) and exposed to NRC-03 in the absence or presence
   of other test compounds. The cells were subsequently incubated for 3 h
   at 37 °C and the absorbance was measured at 450 nm using Multiskan™ FC
   Microplate Photometer (Thermo Scientific).

2.6. Colocalization analysis

   A confocal laser scanning microscopy (CLSM, Leica TCS SP8) was
   performed to observe the subcellular localization of NRC-03.
   CAL-27 cells were seeded confocal dishes at a density of
   2 × 10^5 cells/well. After incubation for 24 h, the cells were treated
   with TRITC-labeled NRC-03 at predetermined intervals, cells were washed
   twice with cold PBS and then incubated with Mitotracker green
   (excitation: 488 nm, emission: 530 nm), LysoTracker Green (excitation:
   504 nm, emission: 511 nm) or Hoechst 33342 (excitation: 350 nm,
   emission: 405 nm) according to the manufacturer's instructions,
   respectively.

2.7. Transmission electron microscope (TEM) analysis

   CAL-27 cells were fixed overnight in 2.5% glutaraldehyde in phosphate
   buffer (0.1 M, pH7.0). After post-fixation with OsO[4] in phosphate
   buffer (0.1 M, pH7.0), cells were dehydrated with a graded series of
   ethanol and embedded in resin. Ultra-thin (70–90 nm) sections were cut,
   stained by uranyl acetate and alkaline lead citrate, and observed in
   TEM (Hitachi H-7650).

2.8. Small interfering RNA (siRNA) transfection

   CypD siRNA targeting human peptidylprolyl isomerase F (PPIF) and
   negative control (NC) were transfected with Lipofectamine 3000 in
   CAL-27 cells according to the manufacturer's instructions. The sequence
   of siRNA-CypD is GACGAGAACTTTACACTGA.

2.9. Measurement of apoptosis by flow cytometry and TUNEL assay

   CAL-27 cells were cultured in 60-mm dishes and treated with or without
   other test compounds for 1 h prior to NRC-03 treatment for 4 h, then
   trypsinized, washed with PBS, and centrifuged at 1000 rpm for 5 min.
   Then, cells were resuspended in 500 μl binding buffer and stained with
   Annexin V-FITC and PI according to the protocol. The cells were
   incubated in the dark at room temperature for 15 min. Finally, the
   percentage of apoptotic cells and necrotic cells were assessed by flow
   cytometer analysis (FACS Aria III Cell Sorter, BD, United States).

   TUNEL staining was carried out to identify the rate of apoptotic cells.
   For the assay, cells inoculated on confocal dishes were fixed in 4%
   paraformaldehyde in PBS and permeabilized with 0.2% Triton X-100 in
   citrate buffer. Samples were incubated with TUNEL reaction mixture at
   37 °C for 1 h, counterstained with Hoechst 33342, and observed with
   CLSM. The percentage of apoptotic cells was estimated by counting a
   total of 300 cells from random fields.

2.10. Caspase activity assay

   CAL-27 cells were pre-treated with or without Z-VAD-FMK for 1 h and
   then co-cultured with NRC-03 for 4 h. Caspase-3 and caspase-8 activity
   were evaluated according to the manufacturer's instructions. Briefly,
   the cell lysate was added to the 96-well plates and incubated with 2 mM
   of the Ac-DEVD-pNA (caspase-3 substrate) or Ac-IETD-pNA (caspase-8
   substrate) at 37 °C overnight. The absorbance was read at 405 nm in
   Multiskan™ FC Microplate Photometer (Thermo Scientific). Protein levels
   in the cell lysate were measured using a Bradford protein assay kit.
   The results were expressed as active units of caspase/μg protein.
   Relative caspase activity was expressed as a fold increase over the
   control.

2.11. Oxygen consumption rate evaluation

   The oxygen consumption rate (OCR) was evaluated using an oxygen
   consumption rate assay kit. Briefly, CAL-27 cells were cultured in a
   96-well plate (8 × 10^4 cells/well) with a clear bottom and black sides
   for 24 h. Next, 100 μl of medium mixed with different concentrations of
   NRC-03 and 5 μl of oxygen fluorescent probe was added to each well.
   Thereafter, blocking buffer (2 drops/well) was immediately added to
   each well to prevent external oxygen generation. After that, the plate
   was read with a fluorescent microplate reader (Model Infinite 200 Pro,
   Tecan) at 37 °C (1 read per 3 min, Ex 455/Em 603). Since the
   fluorescence of this oxygen probe can be quenched by O[2], the value of
   the fluorescence signal was inversely proportional to the amount of
   O[2]in each well. OCR was calculated based on the changes of
   fluorescence signal over 2 h as follows: OCR (%) = (final fluorescence
   in NRC-03-treated cells−initial fluorescence in NRC-03-treated
   cells)/(final fluorescence in control cells−initial fluorescence in
   control cells) × 100%.

2.12. Measurement of cellular oxidative stress

   The determination of intracellular oxidant stress was based on the
   oxidation of DCFH-DA. Briefly, CAL-27 cells were seeded in 60 mm dishes
   and treated with NRC-03 in the absence or presence of other test
   compounds for the indicated time, and then were incubated with
   redox-sensitive dye DCFH-DA at 37 °C for 30 min and analyzed by a flow
   cytometer.

   For determining mitochondrial ROS production, CAL-27 cells were
   cultured in confocal dishes and treated with or without other test
   compounds for 1 h prior to NRC-03 for 4 h. The cells were stained with
   100 nM Mitotracker green for 30 min, and then with 2 μM MitoSOX Red
   (excitation: 510 nm, emission: 580 nm) for 15 min at 37 °C and
   visualized with CLSM. The ratio of fluorescence intensities was
   determined by ImageJ software.

   For determining hydrogen peroxide (H[2]O[2]) production, CAL-27 cells
   were cultured in a 96-well plate (8 × 10^4 cells/well) with a clear
   bottom and black sides for 24 h. The cells were treated with or without
   other test compounds for 1 h prior to NRC-03 treatment for 4 h. The
   quantification of H[2]O[2] production was assessed by AbIR Peroxidase
   Indicator using a Hydrogen peroxide assay kit – (Fluorometric-Near
   Infrared) complemented with a fluorescence plate reader (Model Infinite
   200 Pro, Tecan) at room temperature (Ex 640/Em 680).

2.13. Mitochondrial membrane potential assay

   CAL-27 cells were plated in 60 mm dishes and grown overnight. Cells
   were then treated for 2 h with NRC-03 at the indicated concentrations
   or vehicle control in the absence or presence of other test compounds.
   Cells were then stained with 1 μM JC-1 dye for 20 min at 37 °C, washed,
   and assessed via flow cytometer analysis.

2.14. Measurement of cellular ATP level

   For the measurement of ATP level, whole-cell extracts were lysed in the
   lysis buffer provided in the ATP assay kit. After centrifugation at
   12,000×g for 5 min at 4 °C, the supernatants were transferred to a new
   1.5-ml tube for ATP analysis. The luminescence from a 100 μl sample was
   assayed in a luminometer (Model Infinite 200 Pro, Tecan) together with
   100 μl of ATP detection buffer. A standard curve of ATP concentrations
   (1 nM–1 μM) was prepared from a known amount.

2.15. Western blot analysis

   After the indicated treatments, cells were collected and lysed in cell
   lysis buffer containing a 1% protease inhibitor. Total protein
   concentrations were measured using a BCA protein assay kit. Proteins
   were separated by electrophoresis and transferred to a polyvinylidene
   difluoride (PVDF) membrane. The membranes were blocked with blocking
   buffer and then incubated overnight at 4 °C with the primary antibody,
   followed by incubation with the secondary antibody at room temperature.
   Protein bands were visualized with an enhanced chemiluminescence
   substrate, detected using the Molecular Imager Gel Doc XR + imaging
   system (Bio-Rad, United States), and quantified with Quantity One
   Software.

2.16. RNA sequencing

   The total RNA of CAL-27 cells treated for 4 h with or without NRC-03
   was isolated using an RNeasy mini kit (Qiagen, Germany). Paired-end
   libraries were synthesized by using the TruSeq RNA Sample Preparation
   Kit (Illumina, USA) following TruSeq RNA Sample Preparation Guide.
   Purified libraries were quantified by Qubit 2.0 Fluorometer (Life
   Technologies, USA) and validated by Agilent 2100 bioanalyzer (Agilent
   Technologies, USA) to confirm the insert size and calculate the mole
   concentration. A cluster was generated by cBot with the library diluted
   to 10 pM and then sequenced on the Illumina NovaSeq 6000 (Illumina,
   USA) by Promegen Biotechnology Co., Ltd, Guangzhou, China. The reads
   were aligned with Hisat2 (v 2.1.0) to GRCm38 with default parameters
   [[78]33]. The output SAM (sequencing alignment/map) files were
   converted to BAM (binary alignment/map) files and sorted using SAMtools
   (version 1.3.1) [[79]34]. Gene abundance was expressed as fragments per
   kilobase of exon per million reads mapped (FPKM). StringTie software
   was used to count the fragment within each gene, and the TMM algorithm
   was used for normalization [[80]35]. Differential expression analysis
   for mRNA was performed using R package edgeR. Differentially expressed
   RNAs with |log2(FC)| value > 1 and q value < 0.05, considered as
   significantly modulated, were retained for further analysis.

2.17. RT-qPCR analysis

   To further investigate the effect of NRC-03 on the apoptosis and
   mitochondrial oxidative stress-related gene expression of OSCC cells,
   CAL-27 cells treated for 4 h with or without NRC-03 were collected and
   total RNAs were extracted for RT-qPCR. Following the manufacturer's
   instructions, complementary DNA was synthesized from 500 ng of total
   RNA using a Takara PrimeScript™ RT Master Mix in T100 Thermal Cycler
   (Bio-Rad, United States). The cDNA was assayed using TaKaRa TB Green™
   Premix Ex Taq™ in the CFX96 Real-Time system (Bio-Rad, United States).
   GADPH was used as a reference gene. The primers used for RT-qPCR are
   listed in [81]Supplementary Table S1.

2.18. Animal xenograft model

   Female BALB/c nu/nu mice (5-weeks-old) were purchased from the Center
   for Experimental Animals, Southern Medical University, Guangzhou,
   China. All animal studies were conducted in accordance with the
   guidelines of the National Regulation of China for Care and Use of
   Laboratory Animals (South China Normal University, Guangzhou, China).
   Southern Medical University Experimental Animal Ethics Committee
   approved all animal care and study protocols (L2018153). Mice were
   subcutaneously injected in the right flank with 4 × 10^6 CAL-27 cells
   in 0.1 ml sterile PBS. At 2–4 weeks post-inoculation, tumors grew to an
   average volume of 100 mm^3 and the CAL-27 tumor-bearing mice were
   randomly distributed into two groups (n = 6 per group) and intratumor
   injected with 50 μl PBS vehicle or 125 μg NRC-03 in 50 μl PBS.
   Treatments were repeated every other day for 15 days. Mice were
   monitored daily for tumor growth (using digital calipers), cachexia,
   and weight loss. Tumor volumes were calculated using the elliptical
   formula: 1/2 (length × width^2). Some tumors were frozen in liquid
   nitrogen for western blot and the other part tumors and the organs
   including the heart, liver, spleen, lungs, and kidneys were
   formalin-fixed and processed for histological analysis.

   Hematoxylin and eosin (H&E) staining, Ki-67 staining, and TUNEL
   staining were performed to detect proliferating cells and apoptotic
   cells respectively.

2.19. Statistical analysis

   All data were representative results from at least three independent
   experiments and mean ± SD. Statistical analysis was performed using
   one-way analysis of variance (ANOVA) and unpaired Student's t-test by
   Graphpad Prism 6.0. p < 0.05 was considered statistically significant.

3. Results

3.1. NRC-03 induced apoptosis in oral squamous cell carcinoma cells

   CCK-8 assay showed that NRC-03 in the concentration range of
   15–75 μg/ml inhibited the viability of CAL-27 and SCC-9 cells in a
   time- and dose-dependent manner ([82]Fig. 1a). Cytotoxicity of all the
   tested concentrations of NRC-03 toward HOK cell was minimum compared
   with CAL-27 or SCC-9 cells ([83]Fig. 1a). Annexin/propidium iodide
   staining assay is an indicator of alterations in cell membrane
   permeability and apoptosis. Annexin/propidium iodide staining revealed
   a dose-dependent increase in apoptosis in NRC-03-treated CAL-27 and
   SCC-9 cells. NRC-03 (30–60 μg/ml) significantly enhanced the rate of
   apoptosis in CAL-27 and SCC-9 cells by about 2.8–4.4-fold. In contrast,
   the enhancement magnitude of HOK apoptosis was much lower (about
   1.2–2.3-fold) ([84]Fig. 1b and 1c). The pro-apoptotic effects of NRC-03
   were further verified by TUNEL staining as an indicator of DNA damage
   ([85]Fig. 1d). The percentage of TUNEL positive cells significantly
   increased in OSCC groups, i.e., 31.61% ± 7.98% in CAL-27 and
   28.25% ± 6.64% in SCC-9 cells, respectively ([86]Fig. 1e), while TUNEL
   staining positivity rate was only 2.37% ± 1.90% in HOK cells after
   treatment with 45 μg/ml NRC-03. In contrast, it is important to note
   that NRC-03 did not substantially affect the viability or induce
   apoptosis in the normal HOK cells ([87]Fig. 1a, 1d, and 1e). Caspase-3
   upregulates during apoptosis-mediated cell death. Caspase-8 is a
   crucial initiator in the death receptor-mediated apoptotic pathway.
   Next, we detected the caspase-3 and caspase-8 activity in
   NRC-03-treated CAL-27 cells. NRC-03 treatment didn't activate the
   caspase-8 activity but caused a remarkable increase in caspase-3
   activity of OSCC cells ([88]Fig. 1f and 1g). The median lethal dose of
   NRC-03 treatment was 45 μg/ml on 4 h treatment. NRC-03 at a
   concentration of 45 μg/ml significantly inhibited the cell viability of
   CAL-27 and SCC-9 cells on 24 h treatment, but the effect on normal HOK
   viability was not that prominent ([89]Fig. 1a). Based on these
   findings, 45 μg/ml of NRC-03 was chosen as the optimal dose to treat
   the cells in subsequent experiments.

Fig. 1.

   [90]Fig. 1
   [91]Open in a new tab

   NRC-03 induced apoptosis in oral squamous cell carcinoma cells. (a)
   Cell viability of NRC-03-treated OSCC cells CAL-27 and SCC-9, and HOK
   analyzed by CCK-8 assay (n = 4). NRC-03 treatment for 4 h induced
   apoptosis in OSCC cells. (b) Detection of phosphatidylserine exposure
   by Annexin-V FITC/PI staining and analysis by flow cytometry. Cells in
   Q1, Q2, Q3, and Q4 respectively represent cell debris
   (Annexin-V^-/PI^+), cells in late apoptosis (Annexin-V^+/PI^+), cells
   in early apoptosis (Annexin-V^+/PI^−), and healthy cells
   (Annexin-V^-/PI^−). (c) The quantification of apoptotic cells
   (Annexin-V^+) (n = 3). (d) Representative immunofluorescence images of
   TUNEL staining of cells treated with 45 μg/ml NRC-03. (e)
   Quantification of TUNEL positive cells (n = 6). (f) Caspase-3 activity
   in NRC-03-treated CAL27 cells (n = 3). (g) Caspase-8 activity in
   NRC-03-treated CAL-27 cells (n = 3). Data are presented as mean ± SD.
   Significant difference compared with the control group, *p < 0.05,
   **p < 0.01, ***p < 0.001, and ****p < 0.0001.

   To further confirm the NRC-03-mediated apoptotic pathway in OSCC cells,
   we pretreated CAL-27 cells with Z-VAD-FMK, an irreversible pan-caspase
   inhibitor, along with NRC-03. Z-VAD-FMK treatment counteracted the
   NRC-03-induced cytotoxicity ([92]Fig. 2a) and apoptosis ([93]Fig.
   2b–e). Furthermore, Z-VAD-FMK nullified the promoting effects of NRC-03
   on the caspase-3 activity in CAL-27 cells ([94]Fig. 2f). Collectively,
   these data verified that NRC-03 induced OSCC cell death mainly via the
   intrinsic/mitochondria-mediated apoptosis pathway.

Fig. 2.

   [95]Fig. 2
   [96]Open in a new tab

   Caspase inhibition attenuated NRC-03-induced apoptosis in CAL-27 cells.
   CAL-27 cells were cultured in the presence of 45 μg/ml NRC-03 for 4 h
   with or without caspase inhibitor Z-VAD-FMK (50 μM). (a) Cell viability
   determined by CCK-8 (n = 4). (b) Flow cytometry analysis of annexin-V
   positive cells. (c) Quantification of annexin-V positive cells (n = 3).
   (d, e) TUNEL immunofluorescence staining and quantification of TUNEL
   positive cells (n = 5). (f) Caspase-3 activity (n = 3). Data are
   presented as mean ± SD. Significant difference between the groups,
   **p < 0.01, ***p < 0.001, and ****p < 0.0001.

3.2. NRC-03 inhibits tumor growth in a xenograft model

   NRC-03 at a dose of 125 μg/animal inhibited the growth of
   CAL-27-derived tumors in a subcutaneous ectopic tumor model in nude
   mice ([97]Fig. 3). Importantly, NRC-03 did not cause observable damage
   to vital organs ([98]Fig. S1a) or bodyweight ([99]Fig. S1b). By day 9,
   compared with the vehicle control, NRC-03 treatment induced a
   significant decrease in tumor growth (p < 0.01), which persisted
   throughout the 15-day study period ([100]Fig. 3b). The final average
   tumor volumes in the control and 125 μg NRC-03 treated groups were
   287.18 ± 66.73 mm^3 and 103.17 ± 48.16 mm^3, respectively ([101]Fig.
   3c). Moreover, tumor tissues from the NRC-03-treated group showed a
   reduced cellular density and proliferation rate ([102]Fig. 3d and e)
   and increased apoptosis rate ([103]Fig. 3d and f) compared with the
   control group. These results in vivo are consistent with the results
   from the in-vitro studies, which highlighted that NRC-03 could inhibit
   OSCC growth via inducing apoptosis in cancer cells.

Fig. 3.

   [104]Fig. 3
   [105]Open in a new tab

   NRC-03 inhibited tumor growth in the ectopic tumor model of OSCC.
   CAL-27-derived xenografts were treated with 125 μg NRC-03 every other
   day for 15 days. (a) Gross images of representative tumor tissues on
   day 15. (b) Tumor volume at different time points (n = 6). (c) Final
   tumor volume on day 15 (n = 6). (d) Representative microscopic images
   of tumor tissue sections showing tumor morphology (H&E staining),
   proliferation (Ki-67 immunohistochemistry), and apoptosis (TUNEL
   staining). Quantification of Ki-67-positive cells (e) (n = 6), and
   apoptotic cells (f) (n = 6). Data are presented as mean ± SD.
   Significant difference compared with the respective control group,
   ***p < 0.001, and ****p < 0.0001.

3.3. NRC-03 damaged the cell membrane and specifically entered the cytoplasm
and nucleus of CAL-27 cells

   To investigate whether NRC-03 entered the cytoplasm of OSCC cells,
   CAL-27 or HOK cells were treated with TRITC labeled NRC-03 for 1 h and
   monitored continuously by CLSM. CLSM revealed that TRITC labeled NRC-03
   rapidly entered the cytoplasm of CAL-27 cells ([106]Fig. 4a). The
   dynamic process of NRC-03 entering the cell can be seen in the
   supplementary information (Supplementary Movie 1 and 2). A remarkably
   increased accumulation of NRC-03 was observed at 8–9 min in
   CAL-27 cells, which was 1.56-fold higher than that in HOK cells at the
   same time. With the prolongation of treatment time, the accumulation of
   NRC-03 gradually increased and stabilized at 30 min in CAL-27. However,
   the cellular accumulation of NRC-03 reached its maximum at 17–18 min in
   HOK and then gradually decreased. Moreover, the amount of NRC-03 in
   CAL-27 cells was 3.48-fold higher than in HOK cells at 60 min
   ([107]Fig. 4b). Importantly, NRC-03 localization in the nucleus of
   CAL-27 cells was higher than that in HOK cells. The cell surface of
   CAL-27 showed a higher accumulation of NRC-03 forming many membrane
   blebs as indicated with black arrows ([108]Fig. 4a). Moreover, some
   CAL-27 cells extruded a peptide-bound substance as indicated with white
   arrows ([109]Fig. 4a), suggesting that the peptides cause significant
   membrane damage to CAL-27 cells. Taken together, these results
   demonstrated that NRC-03 selectively targets OSCC cells, causes
   membrane blebbing, and localizes in the nucleus.

Fig. 4.

   [110]Fig. 4
   [111]Open in a new tab

   NRC-03 damaged the cell membrane of CAL-27 cells and entered the
   cytoplasm and nucleus. CLSM images of cells were treated with 45 μg/ml
   NRC-03 for 1 h and photographed by CLSM every 30 s. (a) Peptide and
   nucleus were visualized by using TRITC-NRC-03 (Red) and Hoechst 33342
   (Blue), respectively. The white arrow indicates extruded peptide-bound
   substance from CAL-27 cells. The black arrow indicates membrane blebs
   on the cell surface of CAL-27. (b) Quantification of the mean
   fluorescence intensity of TRITC-NRC-03 at different time points. (For
   interpretation of the references to color in this figure legend, the