Colorectal cancer (CRC) is one of the most common malignancies and the fourth leading cause of cancer death in the world, with nearly 50 000 patients dying each year as a result of metastatis 1. Recent studies have made advances in the development of a cure for CRC, including multimodal and combined therapeutic approaches of surgical resection, radiotherapy, and chemotherapy, to significantly increase the median overall survival 2. Despite these advances, the development of new strategies for CRC is critical. In CRC, many genetic mutations activate tumor development 3. The mutations include those found in oncogenes and antioncogenes, including adenomatous polyposis coli (APC; present in 80% of CRCs), TP53 (50%), KRAS (35–45%), PIK3CA (20–30%), and BRAF (10%) 4. In CRC, the PI3K/AKT signaling plays an important role in CRC development and progression and is a potential therapeutic target for CRC 5–7.
The AKT family contains a serine/threonine protein kinase that is easily stimulated by a variety of extracellular irritants through the phosphatidylinositol 3-kinase pathway that regulates many cellular functions such as cell survival, metabolism, differentiation, and proliferation 8. The mammalian target of rapamycin (mTOR) is a typical Ser/Thr protein kinase, which is a crucial downstream signaling molecule of AKT, and P70S6K and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) are two of its main downstream targets that regulate cellular process such as translation initiation, protein synthesis, cell cycle, and cell migration 9. The PI3K/AKT pathway controls malignancy development in breast cancer, liver cancer, and lung cancer. Furthermore, PI3K/AKT signaling plays a crucial role in cell growth, proliferation, and migration, which are central regulators of CRC 10,11.
Matrix metalloproteinases (MMPs), a family of calcium-dependent zinc-containing endopeptidases, are responsible for extracellular matrix degradation and remodeling of tissues. MMPs are involved in various physiological mechanisms in normal and pathological processes, including embryogenesis, wound healing, inflammation, arthritis, and cancer 12. Ample evidence has confirmed the association between MMPs and tumor growth, migration, and metastasis in CRC, including MMP3 and MMP713. However, to the best of our knowledge, the functional role of MMP3 and MMP7 in the initiation and progression of CRC remains to be elucidated.
Celastrol, a pentacyclic triterpene derived from the traditional Chinese medicinal plant Tripterygium wilfordii Hook F (also known as thunder of god vine), possesses a wide variety of activities for the treatment of chronic inflammation, cancer, and obesity 14–17. Recently, several studies have shown that celastrol can efficiently suppress tumor proliferation, migration, and angiogenesis in multiple tumor models both in vitro and in vivo18–20. Celastrol inhibits proliferation, migration, invasion, and suppresses the CIP2A/c-MYC signaling pathway 17. Celastrol also inhibits ovarian cancer cell migration and invasion through downregulation of the nuclear factor-κB pathway 21. In addition, celastrol has effects on the expression of many oncogenes and tumor-suppression genes, including CXCR4, vascular endothelial growth factor receptor, and CIP2A (the p90 tumor-associated antigen) 22–24. Notably, we found that celastrol can also directly downregulate MMP3 and MMP7 through the PI3K/AKT signaling pathway in CRC cells.
The aim of this study was to investigate the potential antitumor effects of celastrol in CRC. Furthermore, a possible molecular mechanism underlying the effects of celastrol on CRC was assessed to determine whether the PI3K/AKT signaling pathway was involved. A better understanding of the molecular mechanisms of CRC progression and the function of celastrol in CRC prevention may support its use as an agent for the treatment of CRC.
Materials and methods
Cell cultures and celastrol or rapamycin treatment
The human CRC cells SW480 and HCT116 were purchased from the American Type Culture Collection (Rockville, Maryland, USA). The SW480 and HCT116 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mmol/l L-glutamine, 100 U/ml (penicillin–streptomycin), and 10% fetal bovine serum (FBS) in 5% CO2 at 37°C in a constant-temperature incubator. The cells were cultured in six-well cell culture cluster plates with DMEM supplemented with 10% FBS and treated with 0–2 µmol/l celastrol for 24 h. The cells were cultured in six-well cell culture cluster plates with DMEM supplemented with 10% FBS and treated with 0–100 nmol/l rapamycin for 24 h.
Small interfering RNA transfection
The AKT small interfering RNA (siRNA) and control siRNA were obtained from Ribobio (Guangzhou, China). SW480 and HCT116 cells were plated in serum-free DMEM and transfected with AKT siRNA and control siRNA (100 nmol/l) using Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA). After transfection for 6–8 h, the culture medium was changed to DMEM supplemented with 10% FBS from serum-free DMEM.
Quantitative real-time polymerase chain reaction
Total RNA was isolated from SW480 and HCT116 cells using the TRIzol reagent (Invitrogen), and the reverse transcription of first-strand cDNA was performed at 42°C for 15 min and 85°C for 5 s using a TransScript Top Green qPCR supermix (TransGen, Guangzhou, China) according to the manufacturer’s protocol.
Total protein was isolated from the SW480 and HCT116 cells treated with or without celastrol. The cells or CRC tissue samples were lysed in radioimmunoprecipitation assay buffer (Beyotime Biotechnology, Shanghai, China), and the lysates were incubated on ice for 10 min before the supernatants were collected. A wet western blotting system was used, with β-actin antibody as the loading control (Cell Signaling Technology, Beverly, Massachusetts, USA). The Institutional Ethics Committee of Dalian Medical University approved the study. The names of the vendor, catalog number and dilution of all the antibodies are as follows: AKT (4691, 1 : 1000; Cell Signaling Technology), p-AKT(ser473) (4060s, 1 : 1000; Cell Signaling Technology), mTOR (2983s, 1 : 1000; Cell Signaling Technology), p-mTOR(Ser2448) (2971s, 1 : 1000; Cell Signaling Technology), S6 (2217s, 1 : 1000; Cell Signaling Technology), p-S6(Ser240/244) (5364s, 1 : 1000; Cell Signaling Technology) 4E-BP1 (9644s, 1 : 1000; Cell Signaling Technology), p-4E-BP1(Thr37/46) (2855s, 1 :1000; Cell Signaling Technology), MMP3 (SC-6839, 1 : 200; Santa Cruz Biotechnology), and MMP7 (sc-58388, 1 : 500; Santa Cruz Biotechnology, California, USA).
Cell proliferation assay
Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) assay (Beyotime Biotechnology). Briefly, 4000 cells were seeded into 96-well culture plates with 0–2 µmol/l of celastrol. After an attachment period, 100 ml of CCK-8 assay buffer was added to each well for 24 or 48 h. Cell proliferation was measured according to the manufacturer’s protocol.
Colony formation assay
Human CRC cells, SW480 and HCT116 (5000 cells/well), were seeded into 10-cm plates with 0–2 µmol/l celastrol and cultured at 37°C for 14 days. The cells were then fixed with 4% formaldehyde for 10 min, and the fixed cells were stained with Crystals Purple (Merck, Palo Alto, California, USA) for 10 min, and rinsed with water. The total number of colonies with more than 50 cells was counted using a light microscope.
The SW480 and HCT116 cells were treated with 0 or 2 µmol/l of celastrol for 24 h. Then, 2×105 or 4×105 cells were seeded into the upper chamber of a Transwell plate (8-μm pore size; BD Biosciences, San Jose, California, USA). The lower chamber contained DMEM with 20% FBS. After 24 h, the nonmigrating cells were removed with cotton wool, and migrating cells located on the surface of the lower chamber were stained with crystal violet and counted using a microscope (Leica, Wetzlar, Germany). For the AKT siRNA transfection experiments, the SW480 and HCT116 cells were transfected with AKT siRNA before treatment with 0 or 1 µmol/l celastrol for 24 h. After the celastrol treatment, 2×105 or 4×105 cells were seeded into the upper chamber of the Transwell plates and DMEM medium with 20% FBS was added to the lower chambers. The number of invaded cells was selected randomly and counted from five random fields in each well.
Statistical analyses were performed using SPSS, version 16.0 (SPSS Inc., Chicago, Illinois, USA). Values were expressed as the mean±SD. Quantitative data in paired groups were determined using the Student’s t-test. One-way analysis of variance was performed for multiple group comparisons. A P value less than 0.05 indicated significant differences.
MMP3 and MMP7 were upregulated in CRC
To investigate the role of MMP3 and MMP7 in CRC, the expressions of MMP3 and MMP7 were analyzed using the Cancer Genome Atlas database (https://cancergenome.nih.gov/). MMP3 and MMP7 were found to be upregulated in CRC compared with normal colon tissue (Fig. 1a and b). Furthermore, the expression levels of MMP3 and MMP7 were also analyzed using the oncomine database (https://www.oncomine.org). MMP3 and MMP7 were found to be upregulated in CRC compared with normal colon tissue (Fig. 1c and d). We next analyzed the expression levels of MMP3 and MMP7 in a panel of CRC tissues that were matched with noncancerous adjacent colorectal tissue samples. The expression levels of MMP3 and MMP7 were markedly increased in CRC tissues (Fig. 1e). These results suggest that MMP3 and MMP7 play important roles in CRC.
Celastrol suppressed CRC cell proliferation
The chemical structure of celastrol is shown in Fig. 2a. To investigate the potential antitumor effects of celastrol in CRC cells, SW480 and HCT116 cells were treated with 0–2 µmol/l celastrol for 24 or 48 h and cell proliferation was determined. Celastrol significantly inhibited cell proliferation at more than 1 µmol/l celastrol, but at less than 1 µmol/l the drug showed only modest effects (Fig. 2b and c). To further examine the effects of celastrol on CRC cells, SW480 and HCT116 cells were treated with 1 µmol/l celastrol for 14 days, and the colony formation was determined. Celastrol treatment abolished the colony formation ability of CRC cells (Fig. 2e and f). These results demonstrated that celastrol has antiproliferative and anticarcinogenic effects on CRC cells.
Celastrol inhibited CRC cell migration
To further examine the effects of celastrol on CRC cell migration, we performed Transwell assays to examine inhibitory effects of celastrol on cell migration in CRC cells. After 2 µmol/l celastrol treatment, the migration of SW480 and HCT116 cells was significantly suppressed (Fig. 3a and b). These results indicated that celastrol inhibited CRC cell migration.
AKT inhibition suppressed MMP3 and MMP7 expression
Next, we tested whether MMP3 and MMP7 were involved in the PI3K/AKT signaling pathway effects. The siRNAs were used to transfect and block AKT expression in CRC cells. RT-qPCR and western blots were used to measure the efficiency of AKT siRNA and protein expression, respectively. The mRNA expression and protein expression was significantly decreased compared with the control group in SW480 and HCT116 cells (Fig. 4a and b). We then measured the mRNA levels of MMP3 and MMP7 in AKT siRNA and control siRNA-transfected CRC cells. The mRNAs of MMP3 and MMP7 were decreased in the AKT siRNA-transfected cell group (Fig. 4c and d). We also measured the mRNAs of other MMPs (including MMP13 and MMP14), but the expression levels of MMP13 and MMP14 were not affected (Fig. 4e and f). In addition, western blots were used to verify these results. Knockdown of AKT downregulated MMP3 and MMP7 expression in SW480 and HCT116 cells (Fig. 4g). These results suggested that MMP3 and MMP7 functioned downstream of the PI3K/AKT pathway, and were necessary for CRC initiation and progression.
To further investigate whether MMP3 and MMP7 got involved in the PI3K/AKT/mTOR signaling pathway effects, we used rapamycin, the mTOR inhibitor, to treat CRC cells. SW480 and HCT116 cells were treated with 0–100 nmol/l rapamycin for 24 h. Western blots were used to measure the efficiency of rapamycin and protein expression. The expression levels of p-mTOR(Ser2448) and p-S6(Ser240/244) were obviously decreased. However, the expression levels of MMP3 and MMP7 were not affected (Fig. 4j). RT-qPCR also was used to measure the mRNA expression of MMP3 and MMP7. The mRNA expression of MMP3 and MMP7 was not inhibited after rapamycin treatment (Fig. 4h and I). These results demonstrated that PI3K/AKT signaling pathway, not mTOR network, is causally involved in regulating MMP3 and MMP7 expression.
AKT silencing promoted celastrol-induced CRC cell proliferation and migration
To further investigate the antitumor effects of celastrol associated with the PI3K/AKT signaling pathway, SW480 and HCT116 cells were transfected with AKT siRNA and control siRNA, before treatment with 0–2 µmol/l celastrol for 0–48 h. The CCK-8 assay was used to measure CRC cell proliferation. The celastrol-induced antiproliferative effects on SW480 and HCT116 cells were enhanced in the AKT siRNA-transfected cells when compared with cells transfected with control siRNA (Fig. 5a and b). We also analyzed inhibitory effects on cells migration in CRC cells using Transwell assays. The celastrol-induced antimigration on SW480 and HCT116 cells was also promoted in the AKT siRNA-transfected cells compared with cells transfected with control siRNA (Fig. 5c and d). These observations showed that inhibition of the PI3K/AKT pathway determined the celastrol-induced antitumor effects in CRC cells.
Celastrol treatment decreased the expression of the PI3K/AKT/mTOR pathway components, MMP3 and MMP7, in CRC cells
To investigate the functional effects of celastrol in the PI3K/AKT/mTOR pathway in CRC, we treated various human CRC cells with varying concentrations of celastrol. After celastrol treatment, the protein expression levels of PI3K/AKT pathway components were significantly inhibited, especially P-AKT (ser473), P-mTOR (Ser2448), P-S6 (Ser240/244), and P-4E-BP1 (Thr37/46), in SW480 cells. At increased celastrol concentrations, the expression levels of total AKT, mTOR, S6, and 4EBP were slightly decreased (Fig. 6a). We also measured the expression of PI3K/AKT pathway components in HCT116 cells. Celastrol obviously decreased the levels of P-AKT (ser473), P-mTOR (Ser2448), P-S6 (Ser240/244), and P-4E-BP1 (Thr37/46) (Fig. 6b), further demonstrating that it could suppress the expression of PI3K/AKT signaling pathway components.
It is well known that CRC cells produce MMPs to control cancer progression, among which MMP3 and MMP7 are significantly involved. Therefore, we determined whether celastrol treatment inhibited MMP3 and MMP7 expression in CRC cells. After celastrol treatment, the expression levels of MMP3 and MMP7 were inhibited in SW480 and HCT116 cells (Fig. 6c and d). This indicated that celastrol inhibited MMP3 and MMP7 expression in CRC cells.
CRC is an irreversible malignant tumor with high mortality. The current therapeutic strategies include surgical resection, radiotherapy, and chemotherapy 25. However, there is no effective strategy capable of curing CRC or preventing its progressive course. CRC is generally associated with mutations in oncogenes and tumor-suppression genes, including AKT26. AKT, a three serine/threonine protein kinase family (AKT1, AKT2, and AKT3), and is activated by a variety of extracellular stimulatory components that play vital roles in many cellular responses, including cell survival, metabolism, differentiation, and proliferation 27. Studies have suggested that activation of AKT was strongly associated with CRC progression and development through activation of AKT and stimulation of its downstream targets 28.
The PI3K/AKT/mTOR pathway is considered to be one of the most important signaling pathways involved in CRC, and the pathway components could regulate cell proliferation, growth, and survival through phosphorylation of a variety of substrates, such as P-AKT and P-mTOR. The mTOR, S6 (S6 ribosomal protein), and 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1, P4E-BP1) are downstream targets of AKT. The mTOR is a central and key serine/threonine kinase involved in various cell signal pathways that are critical for cellular proliferation and the growth of various tumor cells 29. The translation repressor, 4E-BP1, binds to eIF-4E (eukaryotic translation initiation factor 4E) and inhibits translation, protein synthesis, and proliferation 30. The suppression of phosphorylated AKT (P-AKT) can lead to the inhibition of P-mTOR and P-S6, that control multiple cellular functions such as cell proliferation, growth, and migration. P-AKT is a promising target for therapeutic agents in CRC treatment. Studies have shown that MMP3 and MMP7 levels are increased in CRC tumors of different stages, implying their role in CRC development and progression 31,32. In the present study, celastrol treatment significantly inhibited MMP3 and MMP7 expression. In addition, knockdown of AKT was associated with a decrease in MMP3 and MMP7 expression, indicating a functional connection between MMP3, MMP7, and the PI3K/AKT pathway.
Celastrol has previously attracted attention owing to its significant anticancer effects in multiple animal tumor models, including liver cancer 33, breast cancer 34, and prostate cancer. In addition, celastrol was shown to have anti-invasive effects in preclinical models of CRC 35. However, little is known regarding the molecular mechanism of celastrol action on proliferation and migration. Studies have shown that the PI3K/AKT signaling pathway plays a critical role in CRC progression and development. Celastrol significantly inhibited the expression of P-AKT and its downstream targets. The present study suggested that celastrol has antiproliferation and antimigration effects in CRC cells through the downregulation of PI3K/AKT pathway proteins.
In the present study, we hypothesized that the high expression of P-AKT, phosphorylated mTOR, MMP3, and MMP7 caused shorter OS in CRC. Celastrol treatment inhibited CRC cell proliferation and migration, and the expression of P-AKT, phosphorylated mTOR, and phosphorylated S6 were significantly inhibited with increased celastrol concentrations. The expression levels of MMP3 and MMP7 were also obviously suppressed. The results showed that celastrol suppressed CRC cell proliferation and migration associated with MMP3, MMP7, and the PI3K/AKT pathway.
To further determine the interactions between MMP3, MMP7, and the PI3K/AKT pathway, we used siRNA to knock down AKT expression. The expression levels of MMP3 and MMP7 were also obviously decreased, indicating an additional role of the PI3K/AKT pathway in MMP3 and MMP7 expression. We next investigated the antitumor effects of celastrol related to MMP3 and MMP7 expression by the PI3K/AKT pathway. Following AKT silencing by siRNA, the celastrol-induced inhibition of CRC cells was enhanced. These results demonstrated that celastrol suppressed proliferation, migration, in CRC cells, and that the anticancer effect of celastrol was related with the downregulation of the PI3K/AKT pathway.
The present study emphasized the possible therapeutic role of celastrol, and its mechanism of action, for possible development as a novel antitumor agent. We have shown for the first time that celastrol suppressed CRC cell proliferation and migration by regulating MMP3 and MMP7 expression levels through the PI3K/AKT signaling pathway. Hence, the potential therapeutic uses of celastrol are highly encouraging. Further studies are warranted to determine the efficacy, optimal concentration of anticancer effect of celastrol in vitro and in vivo and celastrol may be regard as a promising chemotherapeutic agent.
This work was supported by the Department of Oncology, First Affiliated Hospital of Dalian Medical University.
Conflicts of interest
There are no conflicts of interest.
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