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British Journal of Cancer logoLink to British Journal of Cancer
. 2022 Oct 13;127(12):2108–2117. doi: 10.1038/s41416-022-02002-2

Corynoxine suppresses pancreatic cancer growth primarily via ROS-p38 mediated cytostatic effects

Chunmei Wen 1,#, Qingqing Ruan 1,#, Zhaofeng Li 2, Xiang Zhou 3, Xuezhi Yang 4, Pingwei Xu 5, Percy David Papa Akuetteh 6, Zheng Xu 1,✉, Jie Deng 1,✉
PMCID: PMC9727079  PMID: 36229578

Abstract

Background

Pancreatic cancer is among the most common malignant tumours, and effective therapeutic strategies are still lacking. While Corynoxine (Cory) can induce autophagy in neuronal cells, it remains unclear whether Cory has anti-tumour activities against pancreatic cancer.

Methods

Two pancreatic cancer cell lines, Patu-8988 and Panc-1, were used. Effects of Cory were evaluated by cell viability analysis, EdU staining, TUNEL assay, colony formation assay, and flow cytometry. Quantitative PCR and Western blot were performed to analyse mRNA and protein levels, respectively. In vivo anti-tumour efficacy of Cory was determined by a xenograft model.

Results

Cory treatment inhibited cell proliferation, induced endoplasmic reticulum (ER) stress, and triggered apoptosis in the pancreatic cancer cell lines. CHOP knockdown-mediated inhibition of ER stress alleviated the Cory-induced apoptosis but showed a limited effect on cell viability. Cory induced cell death partially via promoting reactive oxygen species (ROS) production and activating p38 signalling. Pretreatment with ROS scavenger N-acetylcysteine and p38 inhibitor SB203580 relieved the Cory-induced inhibition on cell growth. Cory remarkably blocked pancreatic tumour growth in vivo.

Conclusions

Cory exerts an anti-tumour effect on pancreatic cancer primarily via ROS-p38-mediated cytostatic effects. Cory may serve as a promising therapeutic agent for pancreatic cancer.

Subject terms: Chemotherapy, Chemotherapy

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is among the most common malignant cancers of digestive tract. As a major histological subtype, PDAC accounts for 90% of all cases of pancreatic cancer. It is estimated that 460,000 people were diagnosed with pancreatic cancer and 430,000 died of the disease in 2018 in the US [1]. While surgical resection remains to be the main therapeutic strategy for pancreatic cancer, most of the patients lose the opportunity of surgical treatment due to late-stage diagnoses [2]. In addition, chemotherapy and radiotherapy have a limited role in improving the survival rate of pancreatic cancer patients [3, 4]. At present, patients with pancreatic cancer generally have a poor clinical outcome because of the lack of effective treatments, tumour metastasis and recurrence, and chemo-resistance [2–4]. Thus, it is highly imperative to develop new pharmacological therapies for pancreatic cancer patients.

Corynoxine (Cory) is a natural compound isolated from the hooks of Uncaria macrophylla Wall (Rubiaceae) [5]. Cory and its enantiomer Corynoxine B (Cory B) were originally identified to enhance autophagy and to be neuroprotective against Parkinson’s disease [5, 6]. Cory was reported to suppress vascular smooth muscle cell proliferation, suggesting a potential in treating vascular diseases [7]. Although there are limited studies on therapeutic effects of Cory on cancers, Cory has been shown to enhance chemosensitivity of resistant hepatic carcinoma cells to doxorubicin and can serve as a multidrug resistance-reversing agent in hepatic carcinoma [8]. It remains to be investigated whether Cory has a biological effect on pancreatic cancer.

Endoplasmic reticulum (ER) stress is an adaptive mechanism related to various stimulus conditions and plays a critical role in cancer. Under chronic stimulation, the unfolded protein response (UPR) induces apoptosis involving multiple signalling molecules, such as activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), C/EBP Homologous Protein (CHOP), and protein kinase R-like ER kinase (PERK) [9]. Recent studies suggest that CHOP signalling triggers apoptosis by regulating the expression of anti-apoptotic and pro-apoptotic genes [10]. ROS is produced during the aerobic metabolism in eukaryotic cells and plays a key role in various physiological and pathological processes. Multiple studies have found that some chemotherapeutic drugs can promote the generation of cellular ROS and thus induce apoptosis in various tumours [11–14]. It has also been suggested that ROS triggers ER stress-mediated apoptosis via activation of JNK/p38 pathway in cancer [15–17]. However, the effects of Cory on ER stress and oxidative stress remain unclear.

In this study, we aimed to understand whether Cory imposes an anti-tumour effect against pancreatic cancer. The associated mechanisms were investigated in aspects of ER stress and oxidative stress.

Materials and methods

Cells, instruments, and software

Patu-8988 and Panc-1 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in DMEM medium (Gibco, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (Gibco) and 100 U/ml penicillin/streptomycin (Gibco) at 37 °C in an incubator with 5% CO2. Instruments and software for measurement and analysis included a plate reader (Varioskan Flash; Thermo Scientific, Waltham, MA, USA), a real-time PCR system (7500 Fast ABI; Applied Biosystems, Foster City, CA, USA), a flow cytometry (FACSVerse™; BD Biosciences, San Jose, CA, USA), a fluorescence microscope (Leica Microsystems, Wetzlar, Germany), the Image J software (version 1.8.0; NIH, Bethesda, MD, USA), the Flow Jo software (version 9.3.3; BD Biosciences), and the GraphPad Prism software (version 8.0.1; San Diego, CA, USA).

CCK-8 assay

Cells were seeded in 96-well plates at a density of 5 × 103 cells/well and cultured overnight. After treatments, the medium was replaced with a mixture containing 100 µl fresh medium and 10 µl CCK-8 solution (Dojindo, Kumamoto, Japan). After 3 h of incubation, the absorbance was read at 450 nm on the plate reader.

Colony formation assay

Cells were seeded in 12-well plates at a density of 250 cells/well and cultured for 4 days. After treatments, cells were cultured in a fresh medium for another 5 days. Cells were then fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 15 min and stained with 0.1% crystal violet (Solarbio). Images were captured and cell clones were counted using the Image J software.

Cell cycle and apoptosis analyses

Cells were seeded in 6-well plates at a density of 5 × 105 cells/well and cultured overnight. After treatments, cells were subjected to cell cycle and apoptosis analyses. For cell cycle analysis, cells were fixed with 1 ml of 70% ethanol at −20 °C overnight and then stained with 50 μg/ml propidium iodide (PI, Solarbio) at 37 °C for 30 min in the dark. For apoptosis analysis, cells were collected, resuspended in 100 μl binding buffer, and stained with 5 μl Annexin V-FITC/7-AAD (BD Biosciences, San Jose, CA, USA) at 37 °C for 15 min. Cell cycle distribution and apoptosis rates were examined by flow cytometry and analysed using the Flow Jo software.

Detection of intracellular ROS, lipid oxidation, Ca2+ level, and labile iron pool (LIP)

Cells were seeded in 6-well plates at a density of 5 × 105 cells/well and cultured overnight. After treatments, cells were subjected to different assays. For determination of intracellular ROS, cells were incubated with DMEM containing 10 μM dichlorodihydrofluorescein diacetate (DCFH-DA; Solaribio) at 37 °C for 20 min. For measurement of lipid oxidation, cells were stained with 2 μM C11-BODIPY 581/591 fluorescent probe (Invitrogen, Carlsbard, CA, USA) in phosphate-buffered saline (PBS) at 37 °C for 30 min. For intracellular Ca2+ levels, cells were stained with 5 μM Fluo-4 AM (Beyotime, Shanghai, China) in PBS at 37 °C for 30 min. For detection of LIP, cells were stained with 0.2 μM calcein acetoxymethyl ester (calcein-AM; MCE, Shanghai, China) in PBS at 37 °C for 15 min. Cells were then washed twice with PBS and incubated in the presence or absence of 100 µM deferiprone at 37 °C for 1 h. The difference between deferiprone presence and absence reflects the amount of LIP. After probe reactions, cells were washed, collected, and subjected to flow cytometry. Data were processed using the Flow Jo software.

Western blot

Whole-cell lysates were prepared in RIPA buffer containing phenylmethylsulfonyl fluoride (Solarbio) and phosphatase inhibitor cocktail (Biomake, Houston, TX, USA). Protein concentrations were determined using a BCA kit (Beyotime). Western blot analysis was performed as previously described [18]. Primary antibodies are listed in Table S1. Anti-mouse or anti-rabbit secondary antibodies (1:2000 dilution) were purchased from Cell Signaling Technology (Danvers, MA, USA). Protein bands were visualised with the ECL detection system (Pierce, Rockford, IL, USA) and analysed by the Image J software.

Quantitative PCR (qPCR)

Total RNA was extracted using a RNA purification kit (Qiagen, Germantown, MD, USA). One μg of total RNA samples were reverse transcribed using the Reverse Transcription Kit (Vazyme, Nanjing, China). The qPCR assay was performed using the SYBR Green Master Mix (Vazyme) in the real-time PCR system. The 2−ΔΔCt method was used to determine the relative gene expression normalised to β-actin. PCR primers are listed in Table S2.

EdU and TUNEL assays

EdU assay was performed using the Click-iT Plus EdU Alexa Fluor® 555 Imaging Kit (Invitrogen). Briefly, cells (5 × 103 cells/well) were seeded into 12-well chamber slides and cultured overnight. Cells were treated with 200 μM Cory for 24 h, followed by culture in complete medium containing 10 μM EdU for 3 h. Thereafter, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized in 0.3% Triton X-100 for 10 min at room temperature. Cells were then stained with the Click Reaction Mixture for 30 min. DAPI (Solarbio) staining was used to visualise the nuclei. Images were captured with the fluorescence microscope and processed using the Image J software. TUNEL assay was performed using the One Step TUNEL Apoptosis Assay Kit (Beyotime). In brief, cells were seeded into 12-well chamber slides at a density of 5 × 103 cells per well and cultured overnight. Thereafter, cells were treated with 200 or 400 μM Cory for 24 h, fixed with 4% paraformaldehyde for 30 min, and permeabilized in 0.5% Triton X-100 for 5 min at room temperature. Then, cells were incubated, respectively, with the TUNEL reaction mixture for 1 h and DAPI (Solarbio) for 5 min at 37 °C in the dark. For TUNEL assay on tissue slides, fixation, permeabilization, and staining reactions were carried out as described above. Images were captured by the fluorescence microscope and processed using the Image J software.

Tumour xenograft models

Six-week-old female BALB/C nude mice were purchased from the Experimental Animal Center of Wenzhou Medical University. Panc-1 cells (5 × 106) were injected subcutaneously into the right flank of the BALB/C nude mice. When tumours reached a volume of 100 mm3, 15 mice were divided into 3 groups (five mice per group, no difference in mean body weight between the groups) and intraperitoneally injected with Cory prepared in 0.5% CMC-Na solution at dosage of 0, 25, and 50 mg/kg body weight, respectively, every day (the control mice were injected with an equal volume of 0.5% CMC-Na solution). Tumour volume and body weight were measured every 2 days. Tumour volume was calculated using the following formula: tumour volume (mm3) = 0.52 × length × (width)2. At day 18, the animals were euthanized, and tumours were surgically removed and collected for Western blot and immunostaining analyses. The study protocol was approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University.

Haematoxylin–eosin (HE) staining and immunostaining

HE staining and immunostaining of tissue sections were performed as previously described with slight modification [19, 20]. In brief, the tumour tissue was dissected and fixed in 4% paraformaldehyde in PBS overnight. After fixation, the tissue was dehydrated, clarified with xylene, embedded in paraffin, and then cut into 5-µm-thick sections. For HE staining, the sections were stained with HE. For immunofluorescence, the sections were incubated with primary antibody against Ki67 (1:200 dilution, Cell Signaling Technology), followed by incubation with Fluor 488-conjugated anti-rabbit IgG (Invitrogen). Nuclei were stained by DAPI (Solarbio). For immunohistochemistry, the sections were incubated with primary antibody (Cell Signaling Technology) against CHOP (1:200), γ-H2AX (1:50), or p-p38 (1:100). Immunoreaction was visualised using 3,3′-diaminobenzidine tetrachloride. Images were captured and processed using the Image J software.

Statistical analysis

Statistical analysis was performed using the GraphPad Prism software. Differences between two groups were analysed by Student’s t test. Comparison between multiple groups was made using one-way analysis of variance. Data were presented as mean ± standard deviation (SD) from at least three independent biological replicates. A p value <0.05 indicated statistical significance.

Results

Cory suppresses PDAC cell proliferation

The chemical structure of Cory is depicted in Fig. 1a. Results of CCK-8 assay showed that Cory treatment led to a dose- and time-dependent reduction in cell viability of Patu-8988 and Panc-1 cells (Fig. 1b). Cory treatment also caused changes in cellular morphology (Fig. S1a) and suppressed colony formation capacity of the PDAC cells (Fig. S1b). In consistency, Cory treatment resulted in a remarkable reduction in the expression of cell proliferation marker Ki67 (Fig. 1c and S1c), and a significant decline in proliferation as suggested by the EdU incorporation assay (Fig. 1d).

Fig. 1. The anti-proliferative effects of Cory on PDAC cells.

Fig. 1

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a The chemical structure of Cory. b The viability of Panc-1 and Patu-8988 cells incubated with various doses of Cory (0, 25, 50, 100, 200, and 400 μM) for 24 h (left) or 200 μM Cory for 12, 24, 48, and 72 h, respectively (right). c Protein expression levels of Ki67, p-CHK1, p-ATR, CDK1, CyclinB1, γ-H2AX, and Cdc25c in Panc-1 and Patu-8988 cells incubated with 100 μM Cory for 24 or 48 h. The protein levels were normalised to GAPDH. d Proliferation rates of Panc-1 and PATU-8988 cells treated with 200 μM Cory for 24 or 48 h. Green, EdU; Blue, DAPI. Scale bar, 50 μm. Proliferation index was expressed as the ratio of (EdU-positive cells)/(the number of nuclei) × 100%. e Cell cycle analysis of Panc-1 and Patu-8988 cells treated with 200 μM Cory for 24 h. Values were calculated based on three independent experiments and presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the control.

We further analysed cell cycle distribution of the Cory-treated PDAC cells. Results showed that Cory treatment led to a decrease in cell proportion at G0/G1 and S phases and an accumulation in cell proportion at G2/M phase (Fig. 1e). Western blot analysis showed decreased expression of CDK1, CyclinB1, and Cdc25C in Cory-treated PDAC cells, which are critical molecules for G2/M cell cycle transition (Figs. 1c and S1f, g). These results confirm that Cory induces a G2/M-phase arrest. Since cell cycle arrest can be induced in response to DNA damage, we further investigated whether Cory induces DNA damage in pancreatic cancer cells. Results showed that Cory treatment increased the expression of phosphorylated H2AX at Ser139 (γ-H2AX), a DNA damage indicator (Figs. 1c and S1f, g). ATR-dependent CHK1 activation has been demonstrated to be implicated in DNA damage-induced G2/M cell cycle arrest. Indeed, increased phosphorylation levels of CHK1 and ATR were observed in the PDAC cells treated with Cory (Figs. 1c and S1d, e).

Cory promotes apoptosis in PDAC cells

Cell apoptosis was analysed using Annexin V/7-AAD staining, TUNEL staining, and Western blot for cleaved Caspase3 and cleaved poly (ADP-ribose) polymerase (PARP). Results showed that Cory administration caused a dose- and time-dependent increase in proportion of the early and late apoptotic cells (Fig. 2a). Compared to the control group, the Cory-treated PDAC cells displayed increased TUNEL staining (Fig. 2b) and elevated levels of cleaved Caspase3 and cleaved PARP (Fig. 2c).

Fig. 2. Cory promotes apoptosis in PDAC cells.

Fig. 2

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a FACS-based evaluation of apoptosis rates of Panc-1 and Patu-8988 cells incubated with 200 μM Cory for 24 and 48 h or various concentrations of Cory (0, 200, and 400 μM) for 24 h. b TUNEL assay-based evaluation of apoptosis rates. Green, TUNEL; Blue, DAPI. Scale bar, 50 μm. Apoptosis index was expressed as the ratio of (TUNEL-positive cells)/(the number of nuclei) × 100%. c Protein expression levels of cleaved PARP and cleaved Caspase3 in the cells treated with 100 μM Cory for 24 or 48 h. The protein levels were normalised to GAPDH. Values were calculated based on three independent experiments and presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the control.

ER stress contributes to Cory-induced apoptotic effect but limitedly to cytostatic effect

Results of qPCR showed that the mRNA levels of CHOP, ATF3, ATF4, ATF6, glucose-regulate protein 78 (GRP78), and X-box binding proteins 1 (XBP1) were elevated in the Cory-treated PDAC cells (Fig. S2a). Further Western blot analysis showed that Cory treatment increased the protein levels of GRP78, ATF4, ATF6, CHOP, p-EIF2α, p-PERK, p-IRE1, and p-JNK, but decreased the level of Caspase12 in the cells (Figs. 3a and S2b–g). Previous studies have shown that Ca2+ homoeostasis is altered under ER stress [21], and ROS is implicated in cellular activities related to ER stress [22, 23]. Results of the Fluo4-AM staining and the DCFH-DA probe, respectively, showed that Cory treatment increased intracellular Ca2+ levels (Fig. 3b) and elevated intracellular ROS levels (Fig. 3c) in the PDAC cells.

Fig. 3. Cory promotes ER stress- and CHOP-dependent apoptosis in PDAC cells.

Fig. 3

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a Protein expression levels of p-PERK, GRP78, p-EIF2α, p-JNK1/2, p-IRE1, ATF4, CHOP, Caspase12 and ATF6 in PDAC cells incubated with 100 μM Cory for 0, 3, 6, 12, 24, and 48 h, respectively. The protein levels were normalised to GAPDH. b Fluo4-AM staining-based detection of intracellular Ca2+ levels in the cells treated with various concentrations of Cory (0, 200, 300, and 400 μM) for 24 h. c DCFH-DA-based detection of ROS levels in the cells incubated with 200 μM Cory for 24 h. d Protein expression level of CHOP in siCHOP-transfected cells. The protein level was normalised to GAPDH. e The viability of siCHOP-transfected cells. f FACS-based evaluation of apoptosis rates in siCHOP-transfected cells. g Protein expression levels of cleaved Caspase3, cleaved PARP, and Ki67. The protein levels were normalised to β-actin. CHOP-siRNA-transfected PDAC cells were stimulated with Cory (200 μM) for 24 h (e–g). Values were calculated based on three independent experiments and presented as mean ± SD. *p < 0.05, **p < 0.01, ****p < 0.0001, compared with the control. ####p < 0.0001, compared with the Cory group.

To determine the role of ER stress in the Cory-induced growth inhibition, we performed siRNA-mediated silencing of CHOP in Panc-1 and Patu-8988 cells (Fig. 3d). Using CCK-8 assay and Annexin V-FITC/7-AAD staining, we found that CHOP knockdown per se caused no change in cell viability and apoptosis in the PDAC cells (Figs. 3e, f and S3e). CHOP knockdown mildly restored the Cory-induced changes in cell viability, proliferation, and Ki67 expression (Fig. 3e, g and S3c, d). In contrast, CHOP knockdown moderately reversed the Cory-induced apoptosis (Figs. 3f and S3e) and substantially suppressed the Cory-induced up-regulation of cleaved Caspase3 and cleaved PARP (Figs. 3g and S3a, b).

ER stress is documented to involve in ferroptosis [24, 25], we thus examined the expression of ferroptosis indicators in the PDAC cells. Results showed that Cory treatment decreased the expression of SLC7A11, GPX4, and NCOA4 and increased the expression of FTH1 (Fig. S4a). Both intracellular Fe2+ levels and lipid ROS production were significantly elevated in Panc-1 and PATU-8988 cells treated with Cory (Fig. S4b, c). However, incubation with ferroptosis inhibitor ferrostatin-1 (Fer-1) or desferoxamine (DFO) did not block the Cory-induced cell death (Fig. S4d). In comparison, the drugs could inhibit the erastin-induced ferroptosis (Fig. S4e). These data indicate that ferroptosis may not play a role in the Cory-induced cell death.

ROS is implicated in the Cory-induced cell death

To understand the role of ROS in the Cory-induced ER stress and apoptosis, we treated Panc-1 and Patu-8988 cells with an antioxidant, N-acetylcysteine (NAC). As expected, NAC treatment markedly diminished the Cory-induced ROS production in the PDAC cells (Fig. 4a). For the Cory-induced responses in the PDAC cells, NAC moderately reversed the reduction in cell viability (Fig. 4b), while appeared to have bigger effect on colony formation (Fig. S5a), cell proliferation (Fig. 4c), and apoptosis rate (Fig. 4d). In addition, NAC administration differentially inhibited the Cory-induced up-regulation of cleaved Caspase3, cleaved PARP, GRP78, and CHOP in the PDAC cells (Figs. 4e and S5b–f). NAC treatment also blocked the Cory-induced activation of p38, a downstream mediator in response to ROS (Figs. 4e and S5f), and reduced the Cory-induced cytosolic Ca2+ levels (Fig. 4f). These results suggest that ROS may be upstream of ER stress and have a differential impact on the Cory-induced responses.

Fig. 4. Cory induces apoptosis via ROS-dependent ER stress in PDAC cells.

Fig. 4

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a ROS levels of Panc-1 and Patu-8988 cells incubated with NAC (10 mM) for 2 h, followed by 200 μM Cory for 24 h. b The viability of the cells incubated with NAC (10 mM) for 2 h, followed by various concentrations of Cory (0, 25, 50, 100, 200, and 400 μM) for 24 h. c Proliferation rates of the cells incubated with NAC (10 mM) for 2 h, followed by 200 μM Cory for 24 h. Green, EdU; Blue, DAPI. Scale bar, 50 μm. Proliferation index was expressed as the ratio of (EdU-positive cells)/(the number of nuclei) × 100%. d FACS-based analysis of apoptosis. e Protein expression levels of GRP78, p-p38, cleaved Caspase3, CHOP, and cleaved PARP. The protein levels were normalised to β-actin. 1: control, 2: Cory 12 h, 3: Cory 24 h, 4: NAC, 5: NAC + Cory 12 h, and 6: NAC + Cory 24 h. f Detection of intracellular Ca2+ levels by Fluo4-AM staining-based flow cytometry. Values were calculated based on three independent experiments and presented as mean ± SD. **p < 0.01, ****p < 0.0001, compared with the control. ####p < 0.0001, compared with the Cory group.

p38 is involved in the Cory-induced growth inhibition and apoptosis

To examine whether p38 plays a role in the Cory-induced proliferative inhibition and apoptosis, we treated Panc-1 and PATU-8988 cells with SB203580, a p38 inhibitor. Results of CCK-8 (Fig. 5a), colony formation (Fig. S6a), EdU incorporation (Fig. 5b), and apoptosis rate assay (Fig. 5c) showed that SB203580 treatment partially rescued the Cory-induced proliferative inhibition and apoptosis. SB203580 treatment also attenuated the Cory-induced increase in cleaved Caspase3, cleaved PARP, GRP78, and CHOP in the PDAC cells (Figs. 5d and S6b–e). In addition, SB203580 treatment alleviated the Cory-induced Ca2+ release in the cells (Fig. 5e). These results suggest that p38 activation involves in the regulation of Cory-triggered responses.

Fig. 5. Cory induces ROS-dependent apoptosis through p38 activation in PDAC cells.

Fig. 5

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a The viability of Panc-1 and Patu-8988 cells incubated with SB203580 (10 μM) for 2 h, followed by various concentrations of Cory (0, 25, 50, 100, 200, and 400 μM) for 24 h. b Apoptosis detection by Annexin V/7-AAD staining-based FACS analysis. c Proliferation rates of the cells incubated with SB203580 (10 μM) for 2 h, followed by 200 μM Cory for 24 h. Green, EdU; Blue, DAPI. Scale bar, 50 μm. Proliferation index was expressed as the ratio of (EdU-positive cells)/(the number of nuclei) × 100%. d Protein expression levels of GRP78, cleaved Caspase3, CHOP, and cleaved PARP. The protein levels were normalised to β-actin. 1: control, 2: Cory 12 h, 3: Cory 24 h, 4: SB, 5: SB + Cory 12 h, 6: SB + Cory 24 h. e Detection of intracellular Ca2+ levels by Fluo4-AM staining-based flow cytometry. Values were calculated based on three independent experiments and presented as mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the control. ####p < 0.0001, compared with the Cory group.

Cory suppresses PDAC tumour growth in vivo

To characterise the in vivo anti-tumour effect of Cory, we generated nude mice bearing Panc-1 cell xenografts. Results showed that tumour volume was apparently reduced in mice treated with Cory at dosages of 25 and 50 mg/kg body weight compared to the vehicle-treated group (Fig. 6a–c). In addition, tumour weight showed a reduction in the Cory-administered mice (Fig. 6d), while the body weight remains unchanged between the Cory- and vehicle-treated groups (Fig. 6e). Cory treatment destructed tumour tissues as suggested by HE staining (Fig. 6f) and induced ER stress, DNA damage, and p38 activation as indicated by the immunohistochemical staining of CHOP, γ-H2AX, and p-p38, respectively (Figs. 6f and S7a). A decrease of Ki67-positive cells (Figs. 6g and S7b) and an increase of TUNEL-positive cells (Figs. 6h and S7c) were observed in the Cory-treated mice. Results of Western blot using the tissues showed similar expression changes of cleaved Caspase3, cleaved PARP, Ki67, GRP78, CHOP, p-JNK, p-p38, γ-H2AX, CDK1, and CyclinB1 as in the cells (Fig. S7d). These in vivo findings confirm that Cory inhibits PDAC tumour growth and induces ER stress, DNA damage, proliferation arrest, and apoptosis.

Fig. 6. Cory inhibits tumour growth in a xenograft model of PDAC.

Fig. 6

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a Representative images of nude mice with subcutaneous xenograft of pancreatic cancer. n = 5 per group. b Gross appearance of the tumours. c The tumour volumes were measured every 2 days. d Tumour weight was evaluated at day 18. e Body weight was measured every 2 days. f Representative histological examinations of the dissected tumours using HE staining, CHOP, p-p38, and γ-H2AX antibodies. Scale bar, 100 μm. g, h Representative images of Ki67 staining and TUNEL assay on the dissected tumours. Red, Ki67; Green, TUNEL; Blue, DAPI. Scale bar, 50 μm. Proliferation and apoptosis indexes were expressed as the ratio of (Ki67 or TUNEL-positive cells)/(the number of nuclei) × 100%, respectively. Values were calculated based on three independent experiments and presented as mean ± SD. **p < 0.01, ***p < 0.001, compared with the control.

Discussion

UPR is a cellular stress response that is elicited by excessive accumulation of unfolded and misfolded proteins in response to ER stress. Under moderate levels of ER stress, homoeostatic UPR can initiate transcriptional and translational changes that regulate cell survival. In case the homoeostatic processes fail to resolve ER stress, the UPR programme will eventually cause cell death. After being released from GRP78/BiP, three ER stress sensors, PERK, ATF6, and IRE1, initiate their downstream cascades. Previous studies suggest that CHOP could induce cell cycle arrest or apoptosis by regulating multiple pro-apoptotic factors [26, 27]. Moreover, IRE1-mediated activation of JNK1/2 has been shown to promote intrinsic apoptosis by directly phosphorylating c-Jun and inducing p53 expression. Activated c-Jun could induce the expression of Bcl-2 family proteins [28]. It has also been shown that ATF4/CHOP and ATF6/CHOP pathways activate caspase3/9, eventually triggering cell apoptosis [29]. ER stress elicits Ca2+ release from the ER and increases cellular Ca2+ concentration, thus inducing apoptosis in cancer cell lines [30]. By analysing cascades of GRP78/PERK/EIF2α/ATF4/CHOP, GRP78/IRE1/XBP1/CHOP, GRP78/IRE1/JNK, and GRP78/ATF6/CHOP, we demonstrate that Cory treatment induces ER stress in PDAC cells. However, not all the changes occur with the same temporal kinetics and some are not sustained, such that p-PERK and p-IRE1 first increase and then decrease along the time. These phenomena may result from differential responsiveness of different signalling to the Cory-induced toxicity in PDAC cells.

Caspase 12 is an ER-specific caspase involved in regulation of ER stress-mediated apoptosis. IRE1-mediated TRAF2 recruitment causes Caspase12 clustering and activation [9]. However, Cory treatment decreases the Caspase12 level, suggesting that the GRP78/IRE1/TRAF2/Caspase12 cascade may not participate in the Cory-induced apoptosis. The CHOP and its mediated ER stress appear to be responsible for the Cory-induced apoptosis but only mildly affect the Cory-induced growth inhibition. These results suggest that the major contributor to the difference observed in the cell-based assays may not be apoptosis, but rather cell cycle arrest. The CCK-8 assay does not discriminate between cytostatic and apoptotic effects because fewer viable cells can still be a consequence of less proliferation. CHOP knockdown results for the cell viability assay versus the flow cytometry also suggest that apoptosis is only a small component of the overall difference. Therefore, Cory appears to be predominantly cytostatic rather than apoptotic and CHOP does not appear to be the major factor controlling response to Cory. As discussed below, ferroptosis does not contribute to Cory effects and ROS appears to be much more important.

A number of anti-cancer drugs have associated their anti-tumour effect with ROS. Studies have shown that elevated ROS levels can lead to misfolded proteins and ER stress [31, 32]. Isoalantolactone, an active sesquiterpene, promotes apoptosis via ROS-mediated ER stress in prostate cancer cells [33]. Camalexin, an indole phytoalexin, stimulates ROS production, causing ER stress and apoptosis in AML cells [33]. Increased ROS levels can activate the p38 MAPK pathway [34] and induce apoptosis by reducing the expression of anti-apoptotic Bcl-2 and promoting the expression of pro-apoptotic Bax via activation of JNK1/2 and p38 [35–37]. In addition, luteolin, a common dietary flavonoid, triggers ROS-dependent apoptosis through inducing mitochondrial dysfunction and ER stress. NAC could reverse the luteolin-triggered apoptosis, mitochondrial pathway activation, and ER stress through inhibiting ROS production [38]. The present study shows that Cory induces ROS production in PDAC cells, and scavenging ROS by NAC can rescue the Cory-induced cell growth arrest, apoptosis, ER stress, and p38 activation. These findings suggest that ROS is involved in cellular and biological effects of Cory on PDAC cells.

Upon activation, p38 MAPK exerts regulatory effects on cancer development and progression [39, 40]. It has been established that p38 phosphorylation leads to activation of ER stress [41, 42]. The p38-related PERK/EIF2α/ATF4 pathway dictates the shift from autophagy to apoptosis [43]. A recent study showed that ROS induced by the TRPV1 antagonist DWP05195 activates p38, up-regulates CHOP expression, and triggers ER stress-mediated apoptosis in human ovarian cancer cells [17]. Similarly, an anti-HIV compound ilimaquinone was found to sensitise colon cancer cells to TRAIL-induced apoptosis by promoting the expression of death receptors DR4 and DR5 via the ROS-p38-CHOP pathway [44]. In agreement with these findings, a significant up-regulation of p38 phosphorylation is displayed in the Cory-treated PDAC cells. The p38 inhibitor can partially reverse the Cory-induced ER stress, cell apoptosis, and proliferation arrest. Together with the above NAC results, ROS-mediated activation of p38 is important for regulating the anti-tumour effect of Cory.

Elevated ROS has also been shown to be responsible for DNA damage and subsequent cell cycle arrest [45, 46]. Three CDKs and their corresponding cyclins are identified as the master regulators driving the eukaryotic cell cycle at critical steps [47]. For instance, CDK1/Cyclin B facilitates the transition from G2 phase to mitosis [48, 49]. Indeed, Cory inhibits DNA synthesis and induces cell cycle arrest in PDAC cells with reduced expression of Cyclin B1 and CDK1. Cell cycle arrest is often accompanied by activation of DNA damage checkpoints. ATM and ATR, two DNA damage response proteins, are involved in DNA damage response, DNA repair, and apoptosis. Once ATR is activated in response to DNA damage, it phosphorylates its downstream target CHK1, inducing Cdc25C inhibition and subsequent cell cycle arrest at G2/M phase [50]. As a master marker of double-strand breaks, γ-H2AX has been widely used in evaluating genomic instability and DNA damage in cancer cells [51, 52]. In the present study, Cory reduces the expression of Cdc25C and increased the level of p-ATR, p-CHK1, and γ-H2AX, suggesting a promoting effect of Cory on the DNA damage response. Overall, our findings suggest that Cory induces cell cycle arrest in G2/M phase potentially via DNA-damage checkpoint pathways.

Ferroptosis is mainly controlled by the SLC7A11/glutathione/GPX4 axis. Reduction in the level of SLC7A11 and/or GPX4 leads to an accumulation of lipid ROS, which often serves as an indicator of ferroptosis [53]. Activation of NCOA4-mediated ferritinophagy degrades the iron stock protein ferritin, resulting in generation of cellular Fe2+ [54]. Herein, although Cory treatment causes ROS accumulation, Fe2+ elevation, and dysregulation of the ferroptosis indicators, the ferroptosis inhibitors, Fer-1 and DFO, show no effect on the Cory-induced cell death, suggesting that ferroptosis plays a minor role in the Cory-induced responses in PDAC cells.

The xenograft animal study shows no significant reduction in body weight of the Cory-treated mice, suggesting a limited toxicity of Cory in vivo. However, we indeed observed a certain toxicity of Cory to normal pancreatic cells (HPNE and H6C7; data not shown). Since cancer drugs often bring additional destruction to patients, the potential side effect of Cory should be further investigated during future preclinical development. The pancreatic tumour microenvironment plays an important role in therapy responses. While the subcutaneous xenograft of pancreatic cells is an extensively used method and provides an in vivo demonstration for the efficacy of Cory, we have to acknowledge that it would be ideal to use intrapancreatic implantation and move into a mouse model of pancreatic cancer, orthotopic or genetically engineered, to show efficacy in a fully immune competent background. In addition, it would also be important in the future to have an extension of in vitro studies to in vivo to identify the key cellular pathways that mediate the Cory response, such as by xenografting HDAC cells harbouring siRNA for CHOP.

Overall, the present study demonstrates that Cory triggers ER stress-dependent apoptosis and proliferation arrest presumably via ROS-p38 pathway. The suppression of Cory on PDAC growth is primarily via its cytostatic effects. Our findings implicate that Cory has the potential to serve as an effective anti-cancer agent for pancreatic cancer.

Supplementary information

Supplementary Figure Legends (83KB, pdf)
Figure S1 (1.5MB, tif)
Figure S2 (737KB, tif)
Figure S3 (707.9KB, tif)
Figure S4 (1MB, tif)
Figure S5 (549KB, tif)
Figure S6 (470.1KB, tif)
Figure S7 (885.5KB, tif)
Table S1 (16.9KB, docx)
Table S2 (13KB, docx)

Author contributions

JD, ZX, and XY designed the project; CW, QR, and XY performed the molecular biology and cell biology experiments and analysed data. ZL and XZ performed in vivo experiments. JD, ZX, and CW wrote the manuscript. PDPA contributed to the manuscript editing. All authors contributed and approved the final version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (grant number 82102983), Wenzhou Municipal Science and Technology Bureau (grant number Y20210234), and the Fundamental Research Funds for Wenzhou Medical University (grant number KYYW202019).

Data availability

The data of this study are available within the article and in Supplementary Materials.

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

All animal procedures were performed according to the Institutional laboratory animal research guidelines and were approved by Wenzhou Medical University Animal Policy and Welfare Committee. This study conformed to the principles outlined in the Declaration of Helsinki.

Consent for publication

Not applicable.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Chunmei Wen, Qingqing Ruan.

Contributor Information

Zheng Xu, Email: xuz@wmu.edu.cn.

Jie Deng, Email: dengjiewz@163.com.

Supplementary information

The online version contains supplementary material available at 10.1038/s41416-022-02002-2.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure Legends (83KB, pdf)
Figure S1 (1.5MB, tif)
Figure S2 (737KB, tif)
Figure S3 (707.9KB, tif)
Figure S4 (1MB, tif)
Figure S5 (549KB, tif)
Figure S6 (470.1KB, tif)
Figure S7 (885.5KB, tif)
Table S1 (16.9KB, docx)
Table S2 (13KB, docx)

Data Availability Statement

The data of this study are available within the article and in Supplementary Materials.

Articles from British Journal of Cancer are provided here courtesy of Cancer Research UK

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