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Natural product pectolinarigenin exhibits potent anti-metastatic activity in colorectal carcinoma cells in vitro and in vivo
Bioorganic & Medicinal Chemistry 27 (2019) 115089
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Natural product pectolinarigenin exhibits potent anti-metastatic activity in colorectal carcinoma cells in vitro and in vivo
T
Cailing Gana,1, Yali Lib,1, Yan Yua, Xi Yuc, Hongyao Liua, Qianyu Zhangb, Wenya Yinb, ⁎ ⁎ Luoting Yua, , Tinghong Yea, a
Laboratory of Liver Surgery, Oxford University-Sichuan University Gastrointestinal Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China b Department of Nutrition and Food Hygiene, School of Public Health, West China Medical School, Sichuan University, Chengdu, China c Carey Business School, Johns Hopkins University, Baltimore, USA
ARTICLE INFO
ABSTRACT
Keywords: Colorectal cancer Pectolinarigenin Apoptosis Metastasis MDSCs
Colorectal carcinoma (CRC) is one of the most common cancers with high metastatic potential, explaining why identifying new drug candidates that inhibit tumour metastasis is an urgent need. The aim of this study was to evaluate the biological activities of pectolinarigenin (PEC, a natural flavonoid present in Cirsium chanroenicum) in CRC in vitro and in vivo and to determine its underlying mechanism of action. Here, we observed that treatment with PEC could inhibit cell viability and induce apoptosis in cancer cells in a concentration- and timedependent manner. The occurrence of apoptosis was associated with activation of caspase-3 and Bax and decreased expression of Bcl-2. In addition, PEC markedly impaired CRC cell migration and invasion by downregulating the expression of matrix metalloproteinase (MMP-9) and phosphorylated-Stat3Tyr705. Moreover, our studies showed that PEC inhibited abdominal metastasis models of murine colorectal cancer. In addition, histological and immunohistochemical analyses revealed a decrease in Ki67-positive cells, MMP9-positive cells and p-Stat3Tyr705 cells upon treatment with PEC compared to control samples. Furthermore, PEC reduced the number of myeloid-derived suppressor cells (MDSCs) in the blood and tumours, which was accompanied by the increased infiltration of CD8+T cells in the blood. Taken together, our findings suggested that PEC could be used as a natural drug to inhibit CRC metastasis.
1. Introduction Colorectal carcinoma (CRC) is one of the most frequently diagnosed malignancies and is a major cause of cancer-related deaths worldwide.1 According to statistics, approximately 140,250 new CRC cases were detected in the United States in 2018 and an estimated 50,630 patients died from this disease in the same year.2 CRC is among the 5 leading causes of cancer incidence and death in China for both men and women.3 Moreover, the global burden of CRC is expected to increase by 60% to more than 2.2 million new cases and 1.1 million cancer deaths by 2030.4 Although there have been advances in surgical treatments and chemotherapy of CRC, the overall survival percentage has hardly changed in recent years. In addition, approximately 30% of recurrent CRC involves metastasis to liver or lung, and most of these patients have unresectable tumours.5 Therefore, new candidates for metastatic
CRC that exert potential anti-tumour activity and low toxicity are urgently needed. It is widely known that some signalling pathways are involved in CRC progression and metastasis, including signal transduction and activator of transcription 3 (Stat3). Stat3, which has been classified as an oncogene, is constitutively active in many tumours such as colorectal, breast, head and neck, prostate, and chronic lymphocytic leukaemia.6–11 In the case of CRC, Stat3 is involved in the regulation of cell differentiation, proliferation, apoptosis, angiogenesis, metastasis and the immune response.12–14 Moreover, increasing evidence has demonstrated that inhibition of the action of Stat3 could inhibit the growth of CRC in vitro and in vivo.15,16 These data all suggest that the Stat3 pathway may represent a target for therapeutic intervention in CRC. Although many Stat3 inhibitors have been reported, to date no Stat3-targeting drug has been approved by the Food and Drug Administration (FDA).17,18
Corresponding authors at: Laboratory of Liver Surgery, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, 17# 3rd Section, Ren Min South Road, 610041 Chengdu, China. E-mail addresses: [email protected] (L. Yu), [email protected] (T. Ye). 1 Cailing Gan and Yali Li contributed equally to this work. ⁎
https://doi.org/10.1016/j.bmc.2019.115089 Received 5 June 2019; Received in revised form 21 August 2019; Accepted 3 September 2019 Available online 04 September 2019 0968-0896/ © 2019 Elsevier Ltd. All rights reserved.
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Drug discovery is time-consuming, requires a large investment, and is a high-risk process. Natural product derivatives have attracted the attention of researchers and clinicians due to their safety, efficacy and immediate availability and are the best choice for new prodrugs. Pectolinarigenin (PEC), a flavonoid compound, is an active component of the medicinal plant thistle Chromolaena odorata,19 which has been shown to possess numerous biologic functions, such as anti-inflammation, anticancer and anti-allergy activities.20–22 Moreover, PEC could inhibit osteosarcoma growth and metastasis by inhibiting the Stat3 signalling pathway.23 Considering the effects of Stat3 in CRC, we hypothesized that PEC, a potent inhibitor of Stat3, might be useful in the treatment of CRC. To verify this hypothesis, the anti-tumour effects and potential mechanisms of PEC were evaluated in CRC in vitro and in vivo. Our findings showed that PEC could inhibit the viability of CRC cells and impair cell migration and invasion. Importantly, PEC could repress tumour metastasis in the abdominal metastasis model by reducing immunosuppressive cells and enhancing antitumour immunity. In conclusion, these observations implied that PEC might be an effective chemotherapeutic agent against CRC.
proteins Bax, Bcl-2, pro-caspase-3, and cleaved caspase-3 by western blot experiments. The results showed that the expression of cleaved caspase-3 increased in a concentration-dependent manner, pro-caspase3 decreased after treatment with PEC for 48 h (Fig. 2C). Moreover, the expression of Bcl-2 was significantly decreased after treatment with PEC for 48 h, while the expression of Bax and the ratio of Bax/Bcl-2 also increased significantly (Fig. 2C, D), suggesting that PEC-induced apoptosis might occur via the mitochondrial apoptotic pathway. These results indicated that the inhibition of CRC cells by PEC is mediated by the induction of apoptosis. 2.3. PEC impaired cellular migration and invasion Migration and invasion are essential processes for the successful metastasis of tumour cells.25 Therefore, the wound-healing and transwell assays were used to test the effects of PEC on cell migration and invasion. As shown in Fig. 3A, B, the wound-healing assay indicated that PEC inhibited the migration of both CT26 and HCT116 cells in a concentration- and time-dependent manner. Similar results were obtained in transwell migration assays (Fig. 3C, D). Moreover, transwell invasion assays were used to examine the effects of PEC on the invasion of CRC. As shown in Fig. 3C, D, the results showed that PEC could damage CT26 migration and invasion in a concentration-dependent manner. Similar results were observed for HCT116 cells (Fig. 3D). To detect the effects of PEC on CRC migration and invasion at the level of protein, we examined the changes in p-Stat3 and migration-invasionrelated protein MMP-9 after treatment with different concentrations of PEC. The results of western blotting showed that PEC could inhibit the expression of related proteins in a concentration-dependent manner (Fig. 3E, F). Taken together, these results implied that PEC could inhibit the migration and invasion of CRC.
2. Results 2.1. PEC inhibited CRC cell proliferation The constitutive activation of Stat3 plays an important role in CRC and is one of its key pathways. In addition, Stat3 phosphorylation of tyrosine residue 705 (Y705, p-Stat3) is highly expressed in human CRC cell lines.24 To test whether PEC has inhibitory effects on CRC cell lines, we treated three established CRC cell lines with gradient concentrations of PEC from 0 to 40 μM for 24 h, 48 h and 72 h. The results showed that the concentration of PEC inhibited the viability of CRC cell lines in a time-dependent manner (Fig. 1A). To further confirm whether PEC could inhibit CRC viability, a clonogenic assay was conducted after PEC treatment. As shown in Fig. 1B, the clonogenic assay clearly demonstrated that colony formation of HCT116, SW48 and CT26 cells was reduced after exposure to PEC (0–40 μM). In addition, the sizes and numbers of colonies treated with PEC were significantly smaller and less than they were in the negative control group. Taken together, these results indicated that PEC has strong cytostatic and cytotoxic effects on CRC cells.
2.4. Anti-metastatic efficacy of PEC in vivo To determine whether the anti-metastatic activity of PEC in vivo is consistent with its effects in vitro, murine CT26 CRC cells were introduced via ip injection into BALB/C mice to establish the abdominal metastasis model. The mice received the following treatments: vehicle, 25 mg/kg or 50 mg/kg of PEC. Before and after treatment, the abdominal anteroposterior diameter was calculated to determine the abdominal circumference (AC) changes, the tumours in the abdominal cavity were removed and weighed, and tumour nodules were counted. As displayed in Fig. 4A, compared with the vehicle group, administration of PEC (50 mg/kg) resulted in a significant reduction in the weights of tumour nodules. In addition, there was a marked dose-dependent reduction in the number of tumour nodules after treatment with PEC (Fig. 4B). Moreover, compared with the control group, splenomegaly was significantly reduced in the PEC-treated group (Fig. 4C). In addition, compared with the solvent group, the AC of the mice treated with PEC was significantly reduced (Fig. 4D). Immunohistochemistry analyses were performed to evaluate the anti-tumour and anti-metastasis mechanisms of PEC. As shown in Fig. 4E, PEC significantly inhibited the proliferation of nuclear Ki-67-positive cells. We also found that treatment with PEC could inhibit the expression of MMP-9 and p-Stat3 in tumour tissues. Overall, these data suggest that PEC block metastasis, which is consistent with the in vitro data.
2.2. CRC cell apoptosis induced by PEC To examine whether the anti-viability activity of PEC in CRC cells was associated with cell apoptosis, the Hoechst 33258 staining assay was performed to detect apoptosis induced by PEC. As presented in Fig. 1C, Hoechst 33258 staining experiments showed that PEC changed the morphology of HCT116 and CT26 cells and induced apoptosis in a concentration- and time-dependent manner, with bright blue fluorescent condensed nuclei and nuclear fragmentation featured. To further confirm that the apoptosis of HCT116 and CT26 cells was induced by PEC, we also evaluated the percent of apoptosis by FCM using the Annexin V-FITC/PI double labelling technique. As shown in Fig. 2B, the percent of apoptotic cells increased from 4.8% to 8.2% when the concentration was increased from 0 to 40 μM at 24 h. Similar results were observed in HCT116 cells. The apoptosis rate was much higher after 48 h of treatment (Fig. 2A, B). After 48 h, the percentage of apoptotic cells increased from 9.8% to 30.2% when the concentration was increased from 0 to 40 μM, respectively, which is nearly a 20.4% change. As displayed in Fig. 2A, B, treatment of HCT116 cells with PEC for 48 h also resulted in significant dose-dependent apoptosis. Therefore, PEC induced CRC cell apoptosis in a time- and concentration-dependent manner. To further determine the pro-apoptotic effects of PEC on CRC cells at the protein level, we detected the expression of apoptosis-related
2.5. PEC modulated the metastatic environment in vivo Tumour-associated MDSCs (myeloid-derived suppressor cells) have a key role in the development of metastasis.26 Therefore, we examined the number of MDSCs in tumours and blood in vivo, as identified by FCM with CD11b and Gr-1 antibodies. As shown in Fig. 5A, the number of CD11b+/Gr1+ MDSCs was significantly decreased in peripheral blood after treatment with PEC compared with the vehicle group in a concentration-dependent manner. Moreover, we further examined 2
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Fig. 1. PEC reduced viability in CRC cancer cells. (A) Colorectal cell lines CT26, HCT116, and SW48 were treated with different concentrations of PEC for 24, 48 or 72 h and cell viability was measured by the MTT assay. Each point represents the mean ± SD for at least 3 independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001 vs vehicle control). (B) The effects of PEC on colony formation in three CRC cell lines for 12 days; the statistical results of colony formation assays were presented as surviving colonies. Data are expressed as mean ± S.D. from three experiments (*P < 0.05, **P < 0.01 and ***P < 0.001) (C) The fluorescence microscopic (10×) appearance of Hoechst 33258 staining nuclei of CT26 and HCT116 cells with various concentrations of PEC for 48 h; the statistical results of Hoechst 33258 staining are expressed as stained positive cells. Data are expressed as mean ± S.D. from three experiments (*P < 0.05, **P < 0.01 and ***P < 0.001).
tumour-associated MDSC infiltration, and from the FCM data, we found the number of MDSCs also was lower in the tumour after PEC treatment (Fig. 5B). Meanwhile, the blood infiltration of active CD8+ T lymphocytes was increased in the PEC-treated group compared with the control group (Fig. 6). These data suggested that PEC inhibited the infiltration of MDSCs into the tumour and increased the number of CD8+ T lymphocytes, which might be associated with suppression of distant colonization of tumour cells in the CT26 abdominal metastasis model.
continuously activated in CRC and is closely related to its development.24 This suggests that targeting Stat3 may be a potential method for the treatment of CRC. In this study, PEC, a flavonoid compound identified as an inhibitor of Stat3, was evaluated for the first time for its potency against CRC in vitro and in vivo. Our results showed that PEC could inhibit the viability of three types of CRC cells. The MTT assay and clone formation experiments confirmed that PEC inhibited the proliferation and activity of CRC in a concentration- and time-dependent manner. Moreover, in an abdominal metastasis model, fewer cells that were Ki-67-positive were observed in the tumour tissues after PECtreatment than in the control group, indicating that PEC could inhibit cell proliferation both in vitro and in vivo. Apoptotic dysfunction has been recognized as a fundamental component of the pathogenesis of CRC.28,29 Therefore, inducing apoptosis is a therapeutic approach to treat CRC.29 In this work, Hoechst 33258 staining and FCM assays both revealed that PEC treatment induced apoptosis in CRC cells in a timeand concentration-dependent manner. In addition, the occurrence of
3. Discussion CRC is one of the most common malignant tumours seen clinically. The occurrence and development of CRC is a complex pathological process of multi-gene participation and multi-step experiments. Due to its high morbidity and the limits of radiation therapy and chemotherapy, there is an urgent need to discover new potential drug candidates for CRC.27 It has been reported in recent years that Stat3 is 3
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Fig. 2. PEC induces apoptosis in CRC cancer cells. (A) CT26 and HCT116 cells were treated with PEC at indicated doses for 48 h, and the level of apoptosis was evaluated using the Annexin V/PI dual-labelling technique, and determined by FCM. (B) The statistics of apoptotic cells, including the early apoptotic cells (LR, positive for Annexin V only) and the late apoptotic cells (UR, Annexin V and PI-positive), were analysed after various concentrations (0–40 μM) of PEC treatment for 24/48 h. Data are expressed as means SD from three experiments (*p < 0.05; **p < 0.01; ***p < 0.001 vs vehicle control). (C) Western blot analyses of CT26 and HCT116 cells treated (48 h) with different concentrations (0, 10, 20 and 40 μM) of PEC were used to evaluate protein expression of Bax, Bcl-2, pro-caspase-3, and cleaved caspase-3. (D) The percentage of Bax/Bcl-2 ratio was presented in the bar graphs.
apoptosis was associated with the activation of cleaved caspase-3 and Bax and downregulation of Bcl-2, suggesting that PEC-induced apoptosis might occur via the mitochondrial apoptotic pathway. CRC is a type of cancer that can metastasize to surrounding organs.30 Moreover, it has been reported that approximately 30–50% of CRC patients undergoing curative resection subsequently experience local tumour recurrence or metastasis.31 In addition, tumour cell migration and invasion are key steps in the successful metastasis of cancer.32 Therefore, inhibition of this important step is a practical antitumour therapy. Consequently, we used the migration invasion test to verify the effects of PEC on metastasis of CRC. The transwell assays showed that PEC could inhibit the migration and invasion activities of CT26 and HCT116 cells. The breakthrough of basement membranes by tumour cells is a key step in the process of invasion and metastasis of malignant tumours, and matrix metalloproteinases play an important role in this process.33 In addition, constitutive Stat3 also plays an important role in the control of cell migration and invasion.34 Therefore,
we also examined cell invasion and migration-related proteins, such as p-Stat3Tyr705 and MMP-9. Western blot results showed that the expression of MMP-9 was downregulated by PEC in CT26 and HCT116 cells. Moreover, IHC assays results showed that PEC also inhibited the number of MMP-9 and p-Stat3Tyr705 positive metastatic cells. These results suggested PEC inhibited CRC cell migration and invasion, which may occur via the Stat3/MMP-9 pathway. A large amount of data indicates that the tumour microenvironment can promote the occurrence, development, and metastasis of CRC.35–37 MDSCs are key components of the tumour microenvironment that accumulate during tumour formation and promote the immune escape and progression of the heterogeneous cell population.38 In this study, our results showed that treatment of mice with PEC caused a significant reduction in the number of MDSCs in the blood and tumours, which was also accompanied by an increase in CD8+ T cell infiltration in blood. It is therefore conceivable that PEC could enhance anti-tumour effects and inhibit tumour metastasis by downregulating the number of MDSCs. 4
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Fig. 3. PEC inhibits migration and invasion in CRC cancer cells. (A, B) PEC inhibits CT26 and HCT116 migration. Tumour cells were seeded in six-well plates. A ‘wound' was created after the cells grew ~80% confluence. After incubation for 24 h or 48 h the groups were fixed and photographed (10×). The lines indicate the area occupied by the location of the wound, and the migrated cells are quantified. (C, D) Tumour cells were seeded in the top chamber of transwell with serum-free medium, with or without matrigel in the upper chamber, treated with vehicle or various concentrations of PEC. After about 48 h, migrated cells were fixed, stained and photographed (20×) and quantified. (E) Western blot analyses of CT26 and HCT116 cells treated (48 h) with different concentrations (0, 10, 20 and 40 μM) of PEC were used to evaluate protein expression of MMP-9. β-actin served as loading control. (F) Western blot analyses of HCT116 cells treated (48 h) with different concentrations (0, 10, 20 and 40 μM) of PEC were used to evaluate protein expression of Stat3/p-Stat3. β-actin served as loading control.
4. Conclusions
33358 and 0.5% crystal violet were bought from Beyotime (Beijing, China). The primary antibodies against Stat3/P-Stat3Tyr705, MMP-9, pro-caspase-3, cleaved caspase-3, Bax and Bcl-2 were obtained from Cell Signalling Technology (Beverly, MA, USA). β-Actin was purchased from ZSJQ-BIO Co. (Beijing, China). β-Tubulin was purchased from ZENBIO (Chengdu, China). Anti-Ki-67 mouse monoclonal was purchased from Merck Millipore (Billerica, MA, USA). FITC-CD11b-, PEGr1-, FITC-CD8a- and PE-CD69-conjugated antibodies were obtained from BD Biosciences (San Diego, CA, USA). PEC (the structural formula is shown in Supplementary Fig. S1) of 98% purity as measured by HPLC analysis was purchased from Weikeqi Biological Technology Co., Ltd. (Chengdu, Sichuan, China).
In conclusion, this study provides important information regarding the antitumour activities activity of PEC in CRC. To the best of our knowledge, this is the first study to demonstrate the anti-CRC activity of PEC in vitro and in vivo. Mechanistic studies have shown that PEC could inhibit cancer cell growth and induce apoptosis. In addition, we further found that PEC could block cell migration and invasion, impairing CRC cell migration and invasion by downregulating the expression of MMP9 and phosphorylated-Stat3Tyr705. Importantly, PEC could inhibit tumour metastasis by reducing the number of MDSCs cells in the tissue and blood. Moreover, our current studies further indicate that Stat3 activity is important for the response of CRC to PEC and that inhibition of Stat3 by PEC directs the pro-apoptotic activity in tumour cells and positive effects on the tumour immunologic microenvironment. Therefore, these results imply that PEC is a potential therapeutic agent for inhibiting CRC growth and metastasis.
5.2. Cell lines and cell culture The human CRC cell lines, HCT116 and SW48, as well as murine CRC cells CT26 were purchased from the American Type Culture Collection (Rockville, MD, USA). All cells were propagated in RPMI 1640 or DMEM media containing 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% antibiotics (penicillin and streptomycin) in 5% CO2 at 37 °C.
5. Materials and methods 5.1. Materials The Annexin V-FITC Apoptosis Detection Kit was purchased from KeyGen Biotech (Nanjing, China). Dimethyl sulfoxide (DMSO) and 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Hoechst
5.3. Cell viability assay The cell viability of PEC-treated CRC cells was assessed by MTT assay.8 Briefly, the exponentially growing cells (2–6 × 103 cells per 5
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Fig. 4. PEC inhibits proliferation and metastasis of colorectal cancer in vivo. To establish an abdominal metastasis model, a total of 5 × 105 CT26 cells were intraperitoneally injected. On day 6 after post-tumour inoculation, mice were treated with 25 and 50 mg/kg per day of PEC (*P < 0.05, **P < 0.01 and ***P < 0.001 versus vehicle control). (A) Weight of tumour nodules in abdomen metastasis model. (B) Metastatic tumour nodule numbers in abdomen metastasis model. (C) Weight of spleen in abdomen metastasis model. (D) AC was calculated before and after treatment. (E) The immunohistochemical analysis was performed to measure the expressions of Ki67, MMP-9 and p-Stat3 in tumour tissues (20×). P-values for comparison of two groups were determined by TWO-tailed Student’s ttest.
well) were seeded in 96-well plates and incubated for 24 h. Then the cells were treated with different concentrations of PEC (0, 2.5, 5, 10, 20, 40 µM). After treatment for 24 h, 48 h and 72 h, respectively, 20 µl of 5 mg/mL MTT was added to each well and incubated for an additional 2–4 h at 37 °C. The medium was subsequently removed, and the purple-colored precipitates of formazan by the living cells were dissolved in 150 µl of DMSO. The color absorbance was recorded at 570 nm using a Spectra MAX M5 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The data presented are representative of three independent experiments.
48 h, the cells were washed with cold PBS and fixed in 4% paraformaldehyde for 15 min. The cells were stained with Hoechst 33258 solution (5 μg/ml). Then, the nuclear morphology of apoptotic cells was observed by fluorescence microscopy (Leica DM4000B, Leica, Wetzlar, Germany). 5.6. Apoptotic assay To further confirm the apoptosis-inducing effects of PEC, we subsequently estimated the number of apoptotic cells by flow cytometry (FCM). Briefly, colon cancer cells (1–2 × 105 cells/well) were seeded in six-well plates for 24 h. After treatment with various concentrations of PEC for 24 h or 48 h, the cells were harvested and washed with cold PBS twice. The apoptosis levels were examined using an Annexin V-FITC/PI detection kit by FCM. The data were analysed with FlowJo software.
5.4. Colony formation assay In brief, cells were seeded at a specified number (400–800 cells per well) in 6-well plates and treated with various concentrations of PEC (0–40 μM) after 24 h incubation. The fresh medium with or without PEC was changed every three days. After treatment for 12 days, the cells were washed with cold PBS, the colonies were fixed with methanol and stained with a 0.5% crystal violet solution for 15 min, and the colonies (< 50 cells) were counted under a microscope.
5.7. Flow cytometry At the indicated time points, we prepared single-cell suspensions of tumours and blood by mechanic and enzymatic dispersion as described previously.39 Then, 1 × 106 freshly prepared cells were stained with different combinations of fluorochrome-coupled antibodies to CD11b, Gr1, CD69 and CD8. Data were collected by FCM and analysed with FlowJo software.
5.5. Morphological analysis by Hoechst staining To identify whether the PEC-inducing reduction in cell viability was attributable to the apoptosis, we stained the HCT116 and CT26 cells with Hoechst 33258 dye. In brief, HCT116 and CT26 cells (1–2 × 105 cells/well) were plated onto an 18-mm cover glass in a sixwell plate for 24 h. After incubating with different concentrations for
5.8. Western blot analysis The western blot analysis was performed as described previously, 6
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Fig. 5. PEC modulated MDSCs in vivo. (A, B) PEC significantly reduced tumour-associated MDSCs in the abdominal metastasis model. Flow cytometry analysis quantified CD11b+Gr1+ myeloid cells in peripheral blood and tumours after treatment with PEC. Bars show mean ± S.D.; five mice every group (*P < 0.05, **P < 0.01 and ***P < 0.001).
Fig. 6. PEC modulated CD8+ T cells in vivo. Single-cell suspensions prepared from tumours were analysed by flow cytometry for the presence of CD8+CD69+. Bars show mean ± S.D.; five mice every group (*P < 0.05, **P < 0.01 and ***P < 0.001).
with minor modification.40 Briefly, HCT116 and CT26 cells were treated with PEC in different concentration for 48 h, then cells were washed with cold PBS for two times and lysed in RIPA buffer. The protein concentrations were examined using the Lowry method and equalized before loading. Equal amounts of protein from each sample were separated on SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Amersham Bioscience, Piscataway, NJ). Then, the membranes were blocked for 1 h at 37 °C and incubated with specific primary antibodies overnight at 4 °C. After incubation with the relevant secondary antibodies for 1 h, the reactive bands were identified using an enhanced chemiluminescence kit (Amersham). Then, the images were analysed using the Image J computer software (National Institute of Health, Bethesda, MD, USA).
24 h or 48 h incubation, cells were fixed and photographed using a microscope (Zeiss, Jena, Germany). The inhibition percentage for migrating cells in the experimental groups was expressed using 100% as the value assigned for the vehicle group. 5.10. Boyden chamber migration and invasion assay The Boyden chamber (8 μm pore size) migration assay was performed as previously described but with some modifications.39 A total of 1 × 105 cells in 100 μl serum-free medium were added to the upper chamber, and 600 μl of medium containing 10% FBS was added at the bottom. Different concentrations of PEC were added to both chambers. Cells were allowed to migrate for 48 h. Non-migrated cells in the upper chamber were discarded using a cotton swab. The migrated cells were fixed in methanol and stained with 0.5% crystal violet for 20 min. Migrated cells in six randomly selected fields were counted and photographed under a light microscope. The invasion assay was performed according to previous studies. In brief, the upper surface of the transwell membrane was coated with serum-free medium diluted Matrigel (1:5, 60 μl/well, BD Biosciences). After Matrigel polymerization, the
5.9. Wound-healing migration assay The wound-healing migration assay was performed as described previously.40 Briefly, when cancer cells grew to 80% confluence, the cell monolayer was scraped with sterile 0.01 ml pipette tips, and fresh medium containing different concentrations of PEC was added. After 7
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lower compartment of the chambers was filled with 600 μl medium with 10% FBS and 5 × 104 cells in 100 μl serum-free medium were placed in the upper part of each transwell and treated with different concentrations of PEC. After incubation for 48 h, cells on the upper side of the filter were removed. Cells located on the underside of the filter were fixed with methanol and stained with 0.5% crystal violet. Next, migrated cells were counted and photographed under a light microscope. The results were expressed as the percentage inhibition rate of migration compared with the untreated group.
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WP1066, a small molecule inhibitor of the JAK/STAT3 pathway, inhibits ceramide glucosyltransferase activity. Biochem Biophys Res Commun. 2017;491:265–270. [18]. Wei N, Li J, Fang C, et al. Targeting colon cancer with the novel STAT3 inhibitor bruceantinol. Oncogene. 2019. [19]. Wang C, Cheng Y, Liu H, et al. Pectolinarigenin suppresses the tumor growth in nasopharyngeal carcinoma. Cell Physiol Biochem. 2016;39:1795. [20]. Lim H, Son KH, Chang HW, Bae K, Kang SS, Kim HP. Anti-inflammatory activity of pectolinarigenin and pectolinarin isolated from Cirsium chanroenicum. Biol Pharm Bull. 2008;31:2063. [21]. Lee HJ, Venkatarame-Gowda-Saralamma V, Kim SM, et al. Pectolinarigenin induced cell cycle arrest, autophagy, and apoptosis in gastric cancer cell via PI3K/ AKT/mTOR signaling pathway. Nutrients. 2018;10:1043. [22]. Lee S, Lee DH, Kim JC, et al. Pectolinarigenin, an aglycone of pectolinarin, has more potent inhibitory activities on melanogenesis than pectolinarin. Biochem Biophys Res Commun. 2017;493. [23]. Tao Z, Li S, Li J, et al. Natural product pectolinarigenin inhibits osteosarcoma growth and metastasis via SHP-1-mediated STAT3 signaling inhibition. Cell Death Dis. 2016;7:e2421. [24]. Lin L, Liu A, Peng Z, et al. STAT3 is necessary for proliferation and survival in colon cancer-initiating cells. Cancer Res. 2011;71:7226–7237. [25]. Bravo-Cordero JJ, Hodgson L, Condeelis J. Directed cell invasion and migration during metastasis. Curr Opin Cell Biol. 2012;24:277–283. [26]. Hong X, Chunyan Z, Andreas H, Yan D, Robert F, Hua Y. Sunitinib inhibition of Stat3 induces renal cell carcinoma tumor cell apoptosis and reduces immunosuppressive cells. Cancer Res. 2009;69:2506–2513. [27]. Kahouli I, Tomaro-Duchesneau C, Prakash S. Probiotics in colorectal cancer (CRC) with emphasis on mechanisms of action and current perspectives. J Med Microbiol. 2013;62:1107–1123. [28]. Watson AJM. Apoptosis and colorectal cancer. Gut. 2004;53:1701–1709. [29]. Mercier I, Vuolo M, Jasmin JFO, et al. ARC (apoptosis repressor with caspase recruitment domain) is a novel marker of human colon cancer. Cell Cycle. 2008;7:1640–1647. [30]. Matsumura K, Tanaka S, Ito T, et al. Colon cancer with macroscopic invasion into surrounding organs. Gan. 1987;33:49. [31]. Lieberman DA, Winawer SJ, Giardiello FM, Johnson DA, Levin TR. Guidelines for colonoscopy surveillance after screening and polypectomy: a consensus update by the US multi-society task force on colorectal cancer. Gastroenterology. 2012;143:844–857. [32]. Friedl P, Wolf K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer. 2003;3:362–374. [33]. Eissa S, Ali-Labib R, Bassiony MM, Tash F, El-Zayat T. Noninvasive diagnosis of bladder cancer by detection of matrix metalloproteinases (MMP-2 and MMP-9) and their inhibitor (TIMP-2) in urine. Eur Urol. 2007;52:1388–1397. [34]. Tsareva SA, Moriggl R, Corvinus FM, et al. Signal transducer and activator of transcription 3 activation promotes invasive growth of colon carcinomas through
5.11. Mice and tumour models The mice models were as described previously.39 Female BALB/c mice (6–8 weeks old) were obtained from HFK Bioscience CO., LTD., Beijing, China. To establish an abdominal metastasis model, we intraperitoneally (ip) injected female mice with CT26 cells (5 × 105/ 100 μl). On day 6 post-tumour inoculation, mice were randomly distributed into three groups (eight mice in each group) and then received an ip injection of PEC 50 mg/kg, 25mg/kg or vehicle once daily for 14 days. Abdominal circumference and body weight were measured every 3 days. Seeded metastatic tumour nodules in the abdominal cavity were counted by eye and weighed. 5.12. Immunohistochemistry Immunohistochemistry (IHC) staining of tumours sections was described previously.39 Paraffin-embedded tumour sections were stained with primary antibodies (Ki-67, MMP-9 and p-Stat3) using the DAB detection Kit (ZSGB-BIO Co., Beijing, China). 5.13. Statistical analysis Data were represented as mean ± SD of three independent experiments. The two-tailed Student’s t-test was used for statistical analysis and statistically significant P-values were labeled as follows: *P < 0.05; **P < 0.01; ***P < 0.001. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by Key Project of the Science & Technology Department of Sichuan Province (Grant No. 2017JY0071); Graduate Student's Research and Innovation Fund of Sichuan University (2018YJSY110). Compliance with ethical standards Ethical approval: All animal experiments were approved by the Institutional Animal Care and Treatment Committee of Sichuan University in China (New Permit Number: 20171205-3). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmc.2019.115089. References [1]. Ozawa T, Matsuyama T, Toiyama Y, et al. CCAT1 and CCAT2 long noncoding RNAs, located within the 8q.24.21 'gene desert', serve as important prognostic biomarkers in colorectal cancer. Ann Oncol. 2017;28:1882–1888.
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C. Gan, et al. matrix metal loproteinase induction. Neoplasia. 2007;9:279–291. [35]. Joyce JA, Pollard JW. Microenvironmental regulation of metastasis. Nat Rev Cancer. 2009;9:239–252. [36]. Stéphanie G, Jacques H. Role of cancer microenvironment in metastasis: focus on colon cancer. Cancer Microenviron. 2008;1:69. [37]. Tabuso M, Homervanniasinkam S, Adya R, Arasaradnam RP. Role of tissue microenvironment resident adipocytes in colon cancer. World J Gastroenterol. 2017;23:5829. [38]. Lesokhin AM, Hohl TM, Kitano S, et al. Monocytic CCR2(+) myeloid-derived
suppressor cells promote immune escape by limiting activated CD8 T-cell infiltration into the tumor microenvironment. Cancer Res. 2012;72:876–886. [39]. Ye TH, Yang FF, Zhu YX, et al. Inhibition of Stat3 signaling pathway by nifuroxazide improves antitumor immunity and impairs colorectal carcinoma metastasis. Cell Death Dis. 2017;8:e2534. [40]. Zeng A, Hua H, Liu L, Zhao J. Betulinic acid induces apoptosis and inhibits metastasis of human colorectal cancer cells in vitro and in vivo. Bioorg Med Chem. 2019;27:2546–2552.
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Natural product pectolinarigenin exhibits potent anti-metastatic activity in colorectal carcinoma cells in vitro and in vivo
T
Cailing Gana,1, Yali Lib,1, Yan Yua, Xi Yuc, Hongyao Liua, Qianyu Zhangb, Wenya Yinb, ⁎ ⁎ Luoting Yua, , Tinghong Yea, a
Laboratory of Liver Surgery, Oxford University-Sichuan University Gastrointestinal Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu, China b Department of Nutrition and Food Hygiene, School of Public Health, West China Medical School, Sichuan University, Chengdu, China c Carey Business School, Johns Hopkins University, Baltimore, USA
ARTICLE INFO
ABSTRACT
Keywords: Colorectal cancer Pectolinarigenin Apoptosis Metastasis MDSCs
Colorectal carcinoma (CRC) is one of the most common cancers with high metastatic potential, explaining why identifying new drug candidates that inhibit tumour metastasis is an urgent need. The aim of this study was to evaluate the biological activities of pectolinarigenin (PEC, a natural flavonoid present in Cirsium chanroenicum) in CRC in vitro and in vivo and to determine its underlying mechanism of action. Here, we observed that treatment with PEC could inhibit cell viability and induce apoptosis in cancer cells in a concentration- and timedependent manner. The occurrence of apoptosis was associated with activation of caspase-3 and Bax and decreased expression of Bcl-2. In addition, PEC markedly impaired CRC cell migration and invasion by downregulating the expression of matrix metalloproteinase (MMP-9) and phosphorylated-Stat3Tyr705. Moreover, our studies showed that PEC inhibited abdominal metastasis models of murine colorectal cancer. In addition, histological and immunohistochemical analyses revealed a decrease in Ki67-positive cells, MMP9-positive cells and p-Stat3Tyr705 cells upon treatment with PEC compared to control samples. Furthermore, PEC reduced the number of myeloid-derived suppressor cells (MDSCs) in the blood and tumours, which was accompanied by the increased infiltration of CD8+T cells in the blood. Taken together, our findings suggested that PEC could be used as a natural drug to inhibit CRC metastasis.
1. Introduction Colorectal carcinoma (CRC) is one of the most frequently diagnosed malignancies and is a major cause of cancer-related deaths worldwide.1 According to statistics, approximately 140,250 new CRC cases were detected in the United States in 2018 and an estimated 50,630 patients died from this disease in the same year.2 CRC is among the 5 leading causes of cancer incidence and death in China for both men and women.3 Moreover, the global burden of CRC is expected to increase by 60% to more than 2.2 million new cases and 1.1 million cancer deaths by 2030.4 Although there have been advances in surgical treatments and chemotherapy of CRC, the overall survival percentage has hardly changed in recent years. In addition, approximately 30% of recurrent CRC involves metastasis to liver or lung, and most of these patients have unresectable tumours.5 Therefore, new candidates for metastatic
CRC that exert potential anti-tumour activity and low toxicity are urgently needed. It is widely known that some signalling pathways are involved in CRC progression and metastasis, including signal transduction and activator of transcription 3 (Stat3). Stat3, which has been classified as an oncogene, is constitutively active in many tumours such as colorectal, breast, head and neck, prostate, and chronic lymphocytic leukaemia.6–11 In the case of CRC, Stat3 is involved in the regulation of cell differentiation, proliferation, apoptosis, angiogenesis, metastasis and the immune response.12–14 Moreover, increasing evidence has demonstrated that inhibition of the action of Stat3 could inhibit the growth of CRC in vitro and in vivo.15,16 These data all suggest that the Stat3 pathway may represent a target for therapeutic intervention in CRC. Although many Stat3 inhibitors have been reported, to date no Stat3-targeting drug has been approved by the Food and Drug Administration (FDA).17,18
Corresponding authors at: Laboratory of Liver Surgery, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, 17# 3rd Section, Ren Min South Road, 610041 Chengdu, China. E-mail addresses: [email protected] (L. Yu), [email protected] (T. Ye). 1 Cailing Gan and Yali Li contributed equally to this work. ⁎
https://doi.org/10.1016/j.bmc.2019.115089 Received 5 June 2019; Received in revised form 21 August 2019; Accepted 3 September 2019 Available online 04 September 2019 0968-0896/ © 2019 Elsevier Ltd. All rights reserved.
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Drug discovery is time-consuming, requires a large investment, and is a high-risk process. Natural product derivatives have attracted the attention of researchers and clinicians due to their safety, efficacy and immediate availability and are the best choice for new prodrugs. Pectolinarigenin (PEC), a flavonoid compound, is an active component of the medicinal plant thistle Chromolaena odorata,19 which has been shown to possess numerous biologic functions, such as anti-inflammation, anticancer and anti-allergy activities.20–22 Moreover, PEC could inhibit osteosarcoma growth and metastasis by inhibiting the Stat3 signalling pathway.23 Considering the effects of Stat3 in CRC, we hypothesized that PEC, a potent inhibitor of Stat3, might be useful in the treatment of CRC. To verify this hypothesis, the anti-tumour effects and potential mechanisms of PEC were evaluated in CRC in vitro and in vivo. Our findings showed that PEC could inhibit the viability of CRC cells and impair cell migration and invasion. Importantly, PEC could repress tumour metastasis in the abdominal metastasis model by reducing immunosuppressive cells and enhancing antitumour immunity. In conclusion, these observations implied that PEC might be an effective chemotherapeutic agent against CRC.
proteins Bax, Bcl-2, pro-caspase-3, and cleaved caspase-3 by western blot experiments. The results showed that the expression of cleaved caspase-3 increased in a concentration-dependent manner, pro-caspase3 decreased after treatment with PEC for 48 h (Fig. 2C). Moreover, the expression of Bcl-2 was significantly decreased after treatment with PEC for 48 h, while the expression of Bax and the ratio of Bax/Bcl-2 also increased significantly (Fig. 2C, D), suggesting that PEC-induced apoptosis might occur via the mitochondrial apoptotic pathway. These results indicated that the inhibition of CRC cells by PEC is mediated by the induction of apoptosis. 2.3. PEC impaired cellular migration and invasion Migration and invasion are essential processes for the successful metastasis of tumour cells.25 Therefore, the wound-healing and transwell assays were used to test the effects of PEC on cell migration and invasion. As shown in Fig. 3A, B, the wound-healing assay indicated that PEC inhibited the migration of both CT26 and HCT116 cells in a concentration- and time-dependent manner. Similar results were obtained in transwell migration assays (Fig. 3C, D). Moreover, transwell invasion assays were used to examine the effects of PEC on the invasion of CRC. As shown in Fig. 3C, D, the results showed that PEC could damage CT26 migration and invasion in a concentration-dependent manner. Similar results were observed for HCT116 cells (Fig. 3D). To detect the effects of PEC on CRC migration and invasion at the level of protein, we examined the changes in p-Stat3 and migration-invasionrelated protein MMP-9 after treatment with different concentrations of PEC. The results of western blotting showed that PEC could inhibit the expression of related proteins in a concentration-dependent manner (Fig. 3E, F). Taken together, these results implied that PEC could inhibit the migration and invasion of CRC.
2. Results 2.1. PEC inhibited CRC cell proliferation The constitutive activation of Stat3 plays an important role in CRC and is one of its key pathways. In addition, Stat3 phosphorylation of tyrosine residue 705 (Y705, p-Stat3) is highly expressed in human CRC cell lines.24 To test whether PEC has inhibitory effects on CRC cell lines, we treated three established CRC cell lines with gradient concentrations of PEC from 0 to 40 μM for 24 h, 48 h and 72 h. The results showed that the concentration of PEC inhibited the viability of CRC cell lines in a time-dependent manner (Fig. 1A). To further confirm whether PEC could inhibit CRC viability, a clonogenic assay was conducted after PEC treatment. As shown in Fig. 1B, the clonogenic assay clearly demonstrated that colony formation of HCT116, SW48 and CT26 cells was reduced after exposure to PEC (0–40 μM). In addition, the sizes and numbers of colonies treated with PEC were significantly smaller and less than they were in the negative control group. Taken together, these results indicated that PEC has strong cytostatic and cytotoxic effects on CRC cells.
2.4. Anti-metastatic efficacy of PEC in vivo To determine whether the anti-metastatic activity of PEC in vivo is consistent with its effects in vitro, murine CT26 CRC cells were introduced via ip injection into BALB/C mice to establish the abdominal metastasis model. The mice received the following treatments: vehicle, 25 mg/kg or 50 mg/kg of PEC. Before and after treatment, the abdominal anteroposterior diameter was calculated to determine the abdominal circumference (AC) changes, the tumours in the abdominal cavity were removed and weighed, and tumour nodules were counted. As displayed in Fig. 4A, compared with the vehicle group, administration of PEC (50 mg/kg) resulted in a significant reduction in the weights of tumour nodules. In addition, there was a marked dose-dependent reduction in the number of tumour nodules after treatment with PEC (Fig. 4B). Moreover, compared with the control group, splenomegaly was significantly reduced in the PEC-treated group (Fig. 4C). In addition, compared with the solvent group, the AC of the mice treated with PEC was significantly reduced (Fig. 4D). Immunohistochemistry analyses were performed to evaluate the anti-tumour and anti-metastasis mechanisms of PEC. As shown in Fig. 4E, PEC significantly inhibited the proliferation of nuclear Ki-67-positive cells. We also found that treatment with PEC could inhibit the expression of MMP-9 and p-Stat3 in tumour tissues. Overall, these data suggest that PEC block metastasis, which is consistent with the in vitro data.
2.2. CRC cell apoptosis induced by PEC To examine whether the anti-viability activity of PEC in CRC cells was associated with cell apoptosis, the Hoechst 33258 staining assay was performed to detect apoptosis induced by PEC. As presented in Fig. 1C, Hoechst 33258 staining experiments showed that PEC changed the morphology of HCT116 and CT26 cells and induced apoptosis in a concentration- and time-dependent manner, with bright blue fluorescent condensed nuclei and nuclear fragmentation featured. To further confirm that the apoptosis of HCT116 and CT26 cells was induced by PEC, we also evaluated the percent of apoptosis by FCM using the Annexin V-FITC/PI double labelling technique. As shown in Fig. 2B, the percent of apoptotic cells increased from 4.8% to 8.2% when the concentration was increased from 0 to 40 μM at 24 h. Similar results were observed in HCT116 cells. The apoptosis rate was much higher after 48 h of treatment (Fig. 2A, B). After 48 h, the percentage of apoptotic cells increased from 9.8% to 30.2% when the concentration was increased from 0 to 40 μM, respectively, which is nearly a 20.4% change. As displayed in Fig. 2A, B, treatment of HCT116 cells with PEC for 48 h also resulted in significant dose-dependent apoptosis. Therefore, PEC induced CRC cell apoptosis in a time- and concentration-dependent manner. To further determine the pro-apoptotic effects of PEC on CRC cells at the protein level, we detected the expression of apoptosis-related
2.5. PEC modulated the metastatic environment in vivo Tumour-associated MDSCs (myeloid-derived suppressor cells) have a key role in the development of metastasis.26 Therefore, we examined the number of MDSCs in tumours and blood in vivo, as identified by FCM with CD11b and Gr-1 antibodies. As shown in Fig. 5A, the number of CD11b+/Gr1+ MDSCs was significantly decreased in peripheral blood after treatment with PEC compared with the vehicle group in a concentration-dependent manner. Moreover, we further examined 2
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Fig. 1. PEC reduced viability in CRC cancer cells. (A) Colorectal cell lines CT26, HCT116, and SW48 were treated with different concentrations of PEC for 24, 48 or 72 h and cell viability was measured by the MTT assay. Each point represents the mean ± SD for at least 3 independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001 vs vehicle control). (B) The effects of PEC on colony formation in three CRC cell lines for 12 days; the statistical results of colony formation assays were presented as surviving colonies. Data are expressed as mean ± S.D. from three experiments (*P < 0.05, **P < 0.01 and ***P < 0.001) (C) The fluorescence microscopic (10×) appearance of Hoechst 33258 staining nuclei of CT26 and HCT116 cells with various concentrations of PEC for 48 h; the statistical results of Hoechst 33258 staining are expressed as stained positive cells. Data are expressed as mean ± S.D. from three experiments (*P < 0.05, **P < 0.01 and ***P < 0.001).
tumour-associated MDSC infiltration, and from the FCM data, we found the number of MDSCs also was lower in the tumour after PEC treatment (Fig. 5B). Meanwhile, the blood infiltration of active CD8+ T lymphocytes was increased in the PEC-treated group compared with the control group (Fig. 6). These data suggested that PEC inhibited the infiltration of MDSCs into the tumour and increased the number of CD8+ T lymphocytes, which might be associated with suppression of distant colonization of tumour cells in the CT26 abdominal metastasis model.
continuously activated in CRC and is closely related to its development.24 This suggests that targeting Stat3 may be a potential method for the treatment of CRC. In this study, PEC, a flavonoid compound identified as an inhibitor of Stat3, was evaluated for the first time for its potency against CRC in vitro and in vivo. Our results showed that PEC could inhibit the viability of three types of CRC cells. The MTT assay and clone formation experiments confirmed that PEC inhibited the proliferation and activity of CRC in a concentration- and time-dependent manner. Moreover, in an abdominal metastasis model, fewer cells that were Ki-67-positive were observed in the tumour tissues after PECtreatment than in the control group, indicating that PEC could inhibit cell proliferation both in vitro and in vivo. Apoptotic dysfunction has been recognized as a fundamental component of the pathogenesis of CRC.28,29 Therefore, inducing apoptosis is a therapeutic approach to treat CRC.29 In this work, Hoechst 33258 staining and FCM assays both revealed that PEC treatment induced apoptosis in CRC cells in a timeand concentration-dependent manner. In addition, the occurrence of
3. Discussion CRC is one of the most common malignant tumours seen clinically. The occurrence and development of CRC is a complex pathological process of multi-gene participation and multi-step experiments. Due to its high morbidity and the limits of radiation therapy and chemotherapy, there is an urgent need to discover new potential drug candidates for CRC.27 It has been reported in recent years that Stat3 is 3
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Fig. 2. PEC induces apoptosis in CRC cancer cells. (A) CT26 and HCT116 cells were treated with PEC at indicated doses for 48 h, and the level of apoptosis was evaluated using the Annexin V/PI dual-labelling technique, and determined by FCM. (B) The statistics of apoptotic cells, including the early apoptotic cells (LR, positive for Annexin V only) and the late apoptotic cells (UR, Annexin V and PI-positive), were analysed after various concentrations (0–40 μM) of PEC treatment for 24/48 h. Data are expressed as means SD from three experiments (*p < 0.05; **p < 0.01; ***p < 0.001 vs vehicle control). (C) Western blot analyses of CT26 and HCT116 cells treated (48 h) with different concentrations (0, 10, 20 and 40 μM) of PEC were used to evaluate protein expression of Bax, Bcl-2, pro-caspase-3, and cleaved caspase-3. (D) The percentage of Bax/Bcl-2 ratio was presented in the bar graphs.
apoptosis was associated with the activation of cleaved caspase-3 and Bax and downregulation of Bcl-2, suggesting that PEC-induced apoptosis might occur via the mitochondrial apoptotic pathway. CRC is a type of cancer that can metastasize to surrounding organs.30 Moreover, it has been reported that approximately 30–50% of CRC patients undergoing curative resection subsequently experience local tumour recurrence or metastasis.31 In addition, tumour cell migration and invasion are key steps in the successful metastasis of cancer.32 Therefore, inhibition of this important step is a practical antitumour therapy. Consequently, we used the migration invasion test to verify the effects of PEC on metastasis of CRC. The transwell assays showed that PEC could inhibit the migration and invasion activities of CT26 and HCT116 cells. The breakthrough of basement membranes by tumour cells is a key step in the process of invasion and metastasis of malignant tumours, and matrix metalloproteinases play an important role in this process.33 In addition, constitutive Stat3 also plays an important role in the control of cell migration and invasion.34 Therefore,
we also examined cell invasion and migration-related proteins, such as p-Stat3Tyr705 and MMP-9. Western blot results showed that the expression of MMP-9 was downregulated by PEC in CT26 and HCT116 cells. Moreover, IHC assays results showed that PEC also inhibited the number of MMP-9 and p-Stat3Tyr705 positive metastatic cells. These results suggested PEC inhibited CRC cell migration and invasion, which may occur via the Stat3/MMP-9 pathway. A large amount of data indicates that the tumour microenvironment can promote the occurrence, development, and metastasis of CRC.35–37 MDSCs are key components of the tumour microenvironment that accumulate during tumour formation and promote the immune escape and progression of the heterogeneous cell population.38 In this study, our results showed that treatment of mice with PEC caused a significant reduction in the number of MDSCs in the blood and tumours, which was also accompanied by an increase in CD8+ T cell infiltration in blood. It is therefore conceivable that PEC could enhance anti-tumour effects and inhibit tumour metastasis by downregulating the number of MDSCs. 4
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Fig. 3. PEC inhibits migration and invasion in CRC cancer cells. (A, B) PEC inhibits CT26 and HCT116 migration. Tumour cells were seeded in six-well plates. A ‘wound' was created after the cells grew ~80% confluence. After incubation for 24 h or 48 h the groups were fixed and photographed (10×). The lines indicate the area occupied by the location of the wound, and the migrated cells are quantified. (C, D) Tumour cells were seeded in the top chamber of transwell with serum-free medium, with or without matrigel in the upper chamber, treated with vehicle or various concentrations of PEC. After about 48 h, migrated cells were fixed, stained and photographed (20×) and quantified. (E) Western blot analyses of CT26 and HCT116 cells treated (48 h) with different concentrations (0, 10, 20 and 40 μM) of PEC were used to evaluate protein expression of MMP-9. β-actin served as loading control. (F) Western blot analyses of HCT116 cells treated (48 h) with different concentrations (0, 10, 20 and 40 μM) of PEC were used to evaluate protein expression of Stat3/p-Stat3. β-actin served as loading control.
4. Conclusions
33358 and 0.5% crystal violet were bought from Beyotime (Beijing, China). The primary antibodies against Stat3/P-Stat3Tyr705, MMP-9, pro-caspase-3, cleaved caspase-3, Bax and Bcl-2 were obtained from Cell Signalling Technology (Beverly, MA, USA). β-Actin was purchased from ZSJQ-BIO Co. (Beijing, China). β-Tubulin was purchased from ZENBIO (Chengdu, China). Anti-Ki-67 mouse monoclonal was purchased from Merck Millipore (Billerica, MA, USA). FITC-CD11b-, PEGr1-, FITC-CD8a- and PE-CD69-conjugated antibodies were obtained from BD Biosciences (San Diego, CA, USA). PEC (the structural formula is shown in Supplementary Fig. S1) of 98% purity as measured by HPLC analysis was purchased from Weikeqi Biological Technology Co., Ltd. (Chengdu, Sichuan, China).
In conclusion, this study provides important information regarding the antitumour activities activity of PEC in CRC. To the best of our knowledge, this is the first study to demonstrate the anti-CRC activity of PEC in vitro and in vivo. Mechanistic studies have shown that PEC could inhibit cancer cell growth and induce apoptosis. In addition, we further found that PEC could block cell migration and invasion, impairing CRC cell migration and invasion by downregulating the expression of MMP9 and phosphorylated-Stat3Tyr705. Importantly, PEC could inhibit tumour metastasis by reducing the number of MDSCs cells in the tissue and blood. Moreover, our current studies further indicate that Stat3 activity is important for the response of CRC to PEC and that inhibition of Stat3 by PEC directs the pro-apoptotic activity in tumour cells and positive effects on the tumour immunologic microenvironment. Therefore, these results imply that PEC is a potential therapeutic agent for inhibiting CRC growth and metastasis.
5.2. Cell lines and cell culture The human CRC cell lines, HCT116 and SW48, as well as murine CRC cells CT26 were purchased from the American Type Culture Collection (Rockville, MD, USA). All cells were propagated in RPMI 1640 or DMEM media containing 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% antibiotics (penicillin and streptomycin) in 5% CO2 at 37 °C.
5. Materials and methods 5.1. Materials The Annexin V-FITC Apoptosis Detection Kit was purchased from KeyGen Biotech (Nanjing, China). Dimethyl sulfoxide (DMSO) and 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Hoechst
5.3. Cell viability assay The cell viability of PEC-treated CRC cells was assessed by MTT assay.8 Briefly, the exponentially growing cells (2–6 × 103 cells per 5
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Fig. 4. PEC inhibits proliferation and metastasis of colorectal cancer in vivo. To establish an abdominal metastasis model, a total of 5 × 105 CT26 cells were intraperitoneally injected. On day 6 after post-tumour inoculation, mice were treated with 25 and 50 mg/kg per day of PEC (*P < 0.05, **P < 0.01 and ***P < 0.001 versus vehicle control). (A) Weight of tumour nodules in abdomen metastasis model. (B) Metastatic tumour nodule numbers in abdomen metastasis model. (C) Weight of spleen in abdomen metastasis model. (D) AC was calculated before and after treatment. (E) The immunohistochemical analysis was performed to measure the expressions of Ki67, MMP-9 and p-Stat3 in tumour tissues (20×). P-values for comparison of two groups were determined by TWO-tailed Student’s ttest.
well) were seeded in 96-well plates and incubated for 24 h. Then the cells were treated with different concentrations of PEC (0, 2.5, 5, 10, 20, 40 µM). After treatment for 24 h, 48 h and 72 h, respectively, 20 µl of 5 mg/mL MTT was added to each well and incubated for an additional 2–4 h at 37 °C. The medium was subsequently removed, and the purple-colored precipitates of formazan by the living cells were dissolved in 150 µl of DMSO. The color absorbance was recorded at 570 nm using a Spectra MAX M5 microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The data presented are representative of three independent experiments.
48 h, the cells were washed with cold PBS and fixed in 4% paraformaldehyde for 15 min. The cells were stained with Hoechst 33258 solution (5 μg/ml). Then, the nuclear morphology of apoptotic cells was observed by fluorescence microscopy (Leica DM4000B, Leica, Wetzlar, Germany). 5.6. Apoptotic assay To further confirm the apoptosis-inducing effects of PEC, we subsequently estimated the number of apoptotic cells by flow cytometry (FCM). Briefly, colon cancer cells (1–2 × 105 cells/well) were seeded in six-well plates for 24 h. After treatment with various concentrations of PEC for 24 h or 48 h, the cells were harvested and washed with cold PBS twice. The apoptosis levels were examined using an Annexin V-FITC/PI detection kit by FCM. The data were analysed with FlowJo software.
5.4. Colony formation assay In brief, cells were seeded at a specified number (400–800 cells per well) in 6-well plates and treated with various concentrations of PEC (0–40 μM) after 24 h incubation. The fresh medium with or without PEC was changed every three days. After treatment for 12 days, the cells were washed with cold PBS, the colonies were fixed with methanol and stained with a 0.5% crystal violet solution for 15 min, and the colonies (< 50 cells) were counted under a microscope.
5.7. Flow cytometry At the indicated time points, we prepared single-cell suspensions of tumours and blood by mechanic and enzymatic dispersion as described previously.39 Then, 1 × 106 freshly prepared cells were stained with different combinations of fluorochrome-coupled antibodies to CD11b, Gr1, CD69 and CD8. Data were collected by FCM and analysed with FlowJo software.
5.5. Morphological analysis by Hoechst staining To identify whether the PEC-inducing reduction in cell viability was attributable to the apoptosis, we stained the HCT116 and CT26 cells with Hoechst 33258 dye. In brief, HCT116 and CT26 cells (1–2 × 105 cells/well) were plated onto an 18-mm cover glass in a sixwell plate for 24 h. After incubating with different concentrations for
5.8. Western blot analysis The western blot analysis was performed as described previously, 6
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Fig. 5. PEC modulated MDSCs in vivo. (A, B) PEC significantly reduced tumour-associated MDSCs in the abdominal metastasis model. Flow cytometry analysis quantified CD11b+Gr1+ myeloid cells in peripheral blood and tumours after treatment with PEC. Bars show mean ± S.D.; five mice every group (*P < 0.05, **P < 0.01 and ***P < 0.001).
Fig. 6. PEC modulated CD8+ T cells in vivo. Single-cell suspensions prepared from tumours were analysed by flow cytometry for the presence of CD8+CD69+. Bars show mean ± S.D.; five mice every group (*P < 0.05, **P < 0.01 and ***P < 0.001).
with minor modification.40 Briefly, HCT116 and CT26 cells were treated with PEC in different concentration for 48 h, then cells were washed with cold PBS for two times and lysed in RIPA buffer. The protein concentrations were examined using the Lowry method and equalized before loading. Equal amounts of protein from each sample were separated on SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Amersham Bioscience, Piscataway, NJ). Then, the membranes were blocked for 1 h at 37 °C and incubated with specific primary antibodies overnight at 4 °C. After incubation with the relevant secondary antibodies for 1 h, the reactive bands were identified using an enhanced chemiluminescence kit (Amersham). Then, the images were analysed using the Image J computer software (National Institute of Health, Bethesda, MD, USA).
24 h or 48 h incubation, cells were fixed and photographed using a microscope (Zeiss, Jena, Germany). The inhibition percentage for migrating cells in the experimental groups was expressed using 100% as the value assigned for the vehicle group. 5.10. Boyden chamber migration and invasion assay The Boyden chamber (8 μm pore size) migration assay was performed as previously described but with some modifications.39 A total of 1 × 105 cells in 100 μl serum-free medium were added to the upper chamber, and 600 μl of medium containing 10% FBS was added at the bottom. Different concentrations of PEC were added to both chambers. Cells were allowed to migrate for 48 h. Non-migrated cells in the upper chamber were discarded using a cotton swab. The migrated cells were fixed in methanol and stained with 0.5% crystal violet for 20 min. Migrated cells in six randomly selected fields were counted and photographed under a light microscope. The invasion assay was performed according to previous studies. In brief, the upper surface of the transwell membrane was coated with serum-free medium diluted Matrigel (1:5, 60 μl/well, BD Biosciences). After Matrigel polymerization, the
5.9. Wound-healing migration assay The wound-healing migration assay was performed as described previously.40 Briefly, when cancer cells grew to 80% confluence, the cell monolayer was scraped with sterile 0.01 ml pipette tips, and fresh medium containing different concentrations of PEC was added. After 7
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lower compartment of the chambers was filled with 600 μl medium with 10% FBS and 5 × 104 cells in 100 μl serum-free medium were placed in the upper part of each transwell and treated with different concentrations of PEC. After incubation for 48 h, cells on the upper side of the filter were removed. Cells located on the underside of the filter were fixed with methanol and stained with 0.5% crystal violet. Next, migrated cells were counted and photographed under a light microscope. The results were expressed as the percentage inhibition rate of migration compared with the untreated group.
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5.11. Mice and tumour models The mice models were as described previously.39 Female BALB/c mice (6–8 weeks old) were obtained from HFK Bioscience CO., LTD., Beijing, China. To establish an abdominal metastasis model, we intraperitoneally (ip) injected female mice with CT26 cells (5 × 105/ 100 μl). On day 6 post-tumour inoculation, mice were randomly distributed into three groups (eight mice in each group) and then received an ip injection of PEC 50 mg/kg, 25mg/kg or vehicle once daily for 14 days. Abdominal circumference and body weight were measured every 3 days. Seeded metastatic tumour nodules in the abdominal cavity were counted by eye and weighed. 5.12. Immunohistochemistry Immunohistochemistry (IHC) staining of tumours sections was described previously.39 Paraffin-embedded tumour sections were stained with primary antibodies (Ki-67, MMP-9 and p-Stat3) using the DAB detection Kit (ZSGB-BIO Co., Beijing, China). 5.13. Statistical analysis Data were represented as mean ± SD of three independent experiments. The two-tailed Student’s t-test was used for statistical analysis and statistically significant P-values were labeled as follows: *P < 0.05; **P < 0.01; ***P < 0.001. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by Key Project of the Science & Technology Department of Sichuan Province (Grant No. 2017JY0071); Graduate Student's Research and Innovation Fund of Sichuan University (2018YJSY110). Compliance with ethical standards Ethical approval: All animal experiments were approved by the Institutional Animal Care and Treatment Committee of Sichuan University in China (New Permit Number: 20171205-3). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmc.2019.115089. References [1]. Ozawa T, Matsuyama T, Toiyama Y, et al. CCAT1 and CCAT2 long noncoding RNAs, located within the 8q.24.21 'gene desert', serve as important prognostic biomarkers in colorectal cancer. Ann Oncol. 2017;28:1882–1888.
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