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. 2023 Jan 3;37(1):182–189. doi: 10.21873/invivo.13067

Hyperforin Suppresses Oncogenic Kinases and Induces Apoptosis in Colorectal Cancer Cells

LI-CHO HSU 1, CHEN-YU KUO 2, FEI-TING HSU 3, HSIN FENG CHANG 4,#, JING-JIM OU 5,#
PMCID: PMC9843801  PMID: 36593022

Abstract

Background/Aim: Signal transducer and activator of transcription 3 (STAT3), Janus Kinase 1 (JAK1), extracellular signal-regulated kinase (ERK), and protein kinase B (AKT) are essential for malignant transformation and progression in colorectal cancer (CRC) and can be considered as targets for therapeutic interventions. Hyperforin, an active constituent from Hypericum perforatum, has been reported to inhibit inflammation. However, whether hyperforin may suppress CRC progression via inactivation of JAK/STAT3, ERK or AKT signaling remains unclear.

Materials and Methods: Human CRC cells were used to identify the treatment efficacy of hyperforin and its underlying mechanisms of action by MTT, flow cytometry, wound healing, and western blotting assays.

Results: Hyperforin not only induced cytotoxicity, extrinsic/intrinsic apoptosis signaling, but also suppressed the invasion/migration ability of CRC. The phosphorylation of STAT3, JAK1, ERK and AKT was found to be decreased by hyperforin.

Conclusion: Hyperforin inactivates multiple oncogenic kinases and induces apoptosis signaling in CRC cells.

Keywords: Hyperforin, oncogenic kinases, apoptosis, colorectal cancer cells, JAK1, STAT3, ERK, AKT

Colorectal cancer (CRC) is the most common malignancy in both men and women, and the second leading cause of cancer-related death worldwide (1). Poor diet, lack of physical activity, obesity, drinking alcoholic beverages, and cigarette smoking are risk factors for CRC formation (2,3). Current treatment options such as surgery, chemotherapy, radiotherapy, and target therapy are being used for CRC (4). For improving the prognosis of CRC patients, a strategy to develop effective adjuvant therapies potentiating anti-CRC efficacy of current treatment options is of utmost need (5-7).

Exuberant activation of oncogenic kinases and transcription factors such as AKT, extracellular signal-regulated kinase (ERK), signal transducer and activator of transcription 3 (STAT-3), and nuclear factor-kappaB (NF-ĸB) promotes proliferation, survival, and invasion of CRC cells. Suppression of oncogenic kinases and transcription factors contributes to the regression of CRC (8-11). For instance, regorafenib a multikinase inhibitor is used to treat metastatic CRC after the failure of standard chemotherapy. Inactivation of AKT, ERK, and NF-ĸB was involved in the regorafenib-inhibited progression of CRC (12,13).

In recent years, herbal medicine has been recognized as an adjuvant therapy that effectively enhances the therapeutic benefits of chemotherapy and targeted therapy while improving the quality of life in patients with CRC (14,15). Herbal plants and major compounds derived from herbal plants elicit the inhibition of CRC through inducing apoptosis, disrupting cell cycle progression, and blocking oncogenic signaling pathways (16-18). Hyperforin, a bioactive compound isolated from the medicinal plant St. John's wort (Hypericum perforatum), has been shown to inhibit Wnt/β-catenin signaling in CRC cells (19,20). However, anti-CRC effects of hyperforin have not yet been elucidated. The goal of the present study was to investigate the inhibitory effects and mechanisms of action of hyperforin on the survival and invasion of CRC cells.

Materials and Methods

Reagents. (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), hyperforin, dihexyloxacarbocyanine Iodide (DIOC6) and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Reagents FITC-DEVD-FMK (cleaved-caspase-3) (Abcam, Waltham, MA, USA), Red-IETD-FMK (cleaved-caspase-8) (Abcam), FITC-VAD-FMK (cleaved-caspase-9) (Abcam), Fas-L-PE (NOK-1, BioLegend, San Diego, CA, USA), Fas-FITC (DX2, BioLegend), Apoptosis Detection Kit I (BD pharmingen, San Diego, CA, USA) were purchased by different companies as listed.

Cell culture. HT-29 and HCT-116 cells were used as an experimental model. The medium used for cell cultures was RPMI-1640 with 10% fetal bovine serum and 1% penicillin-streptomycin 100x solution. Cells were maintained in an incubator under 37˚C degrees and 5% CO2 atmosphere. All culture-related reagents were purchased from GIBCO®/Invitrogen Life Technologies (Carlsbad, CA, USA) (13).

MTT assay. HT-29 and HCT-116 cells were seeded in 96-well plates (5,000 cells/well) overnight, then, 5, 10, 15, and 20 μM hyperforin were added for 48 h. After 48 h, the medium and cells were incubated at 37˚C for 2 h. Finally, 100 μl DMSO were added before using an ELISA reader to read absorbance at 570 nm.

Flow cytometry analysis for cell cycle and apoptosis-related molecules. HT-29 and HCT-116 cells were seeded in 6 well plates overnight and treated with 5 and 10 μM hyperforin for 48 h. After treatment, cells were digested by trypsin, harvested into 15 ml centrifuge tubes, washed with 1 ml phosphate buffered saline (PBS), fixed with 70% cold ethanol, and then stored at –20˚C overnight. The next day, cells were spun down (2,000 rpm, 10 min), washed with PBS, and stained with propidium iodide in the dark (37˚C for 20 min), and then analyzed by the NovoCyte flow cytometer with the NovoExpress® software (Agilent Technologies Inc., Santa Clara, CA, USA) (21). For apoptosis measurements, cells were stained by cleaved-caspase 3, 8, 9, Fas, Fas-L, DIOC6, and Annexin V-PI reagent in the dark for 30 min at 37˚C (22,23).

Transwell migration and invasion assay. HT-29 and HCT-116 cells were seeded in 6-well plates overnight and treated with 5 and 10 μM hyperforin for 48 h. After treatment, 5×105 cells in 100 μl serum-free medium were loaded onto the upper chamber of transwell (with or without 30% matrigel) and incubated with a bottom chamber 500 μl medium containing 70% FBS. Cells were allowed to invade and migrate for 48 h, followed by the 4% paraformaldehyde fixation and 0.3% crystal violet staining on the transwell membrane. Finally, membranes were collected and observed by a microscope at ×200 magnification (23).

Wound healing assay. HT-29 and HCT-116 cells were pre-treated with 5 and 10 μM hyperforin for 48 h, then cells were seeded in 6 well with ibidi culture inserts (cat: 80241, ibidiGmbH, Gräfelfing, Germany) overnight. The 2-well insert was then removed. Finally, cell the migration pattern was automatically observed by the IncuCyte S3 System (Sartorius, Göttingen, Germany), and images were taken using a IncuCyte S3 system at 0 h, 12 h and 24 h (22).

Western blotting. HT-29 and HCT-116 cells were seeded in 10-cm plates overnight and treated with 5 and 10 μM hyperforin for 48 h. Protein was collected from cells using RIPA buffer. The process was in detail described in previous studies (24). Primary antibodies were purchased from Cell Signaling Technology (CST) (Danvers, MA, USA), including anti-cleaved-caspase 3, 8, 9, anti-BAK, anti-ERK (Thr202/Tyr204), anti-ERK, anti-AKT (Ser473), anti-AKT, anti-PRAS40 (Thr246), anti-PRAS40, anti-JAK (Tyr1034/1035), anti-JAK, anti-STAT3 (Tyr705), anti-STAT3, anti-NF-ĸB (Ser536), anti-NF-ĸB, anti-MCL1, anti-XIAP, anti-CyclinD1 and anti-MMP-9, anti-β-actin and anti-vinculin. After incubation with HRP second antibody for 1 h at 25˚C, we then incubated the membrane with Immobilon Western Chemiluminescent HRP Substrate (Pierce, Rockford, IL, USA) and images were recorded by VisionWorks (Analytik Jena, Jena, Germany). Quantification of data was all performed by loading control vinculin and β-actin.

Results

Hyperforin markedly induced cytotoxicity and apoptosis of CRC cells. Cytotoxicity of HT29 and HCT116 cells was noted after hyperforin treatment for 48 h in a dose-dependent manner (Figure 1A). The IC50 of HT29 and HCT116 was 10.33 μM and 8.73 μM, respectively. Therefore, we used 5 and 10 μM hyperforin to perform the experiments. In Figure 1B, the Annexin-V activation was effectively induced by hyperforin, which indicated that an apoptosis effect was activated in CRC cells. Then, we investigated whether the apoptotic population may also accumulate in CRC after hyperforin treatment. As indicated in Figure 1C, subG1 accumulation was increased to 20-40% by hyperforin in a dose-dependent manner in both CRC cell lines. PARP-1 induced proteolysis of caspases into their cleavage form and thus promoted apoptosis by preventing DNA repair-induced survival (25). Figure 1D and E, shows that hyperforin induced activation of both cleaved caspase-3 and cleaved PARP-1. Taken together, hyperforin-induced CRC cell death is associated with induction of apoptosis pathways.

Figure 1. Cytotoxicity and apoptosis effects of hyperforin on HT29 and HCT116 cells. HT29 and HCT116 cells were treated with different concentrations of hyperforin, and results were evaluated with (A) MTT assay, (B) Annexin-V/PI double staining assay, (C) cell cycle analysis, (D) cleaved caspase-3 staining assay and (E) cleaved PARP-1 staining assay. (*p<0.05, **p<0.01, ***p<0.005 vs. 0 μM hyperforin).

Figure 1

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Hyperforin effectively induced extrinsic and intrinsic apoptosis signaling in CRC cells. To further investigate the detailed mechanism and role of hyperforin on apoptosis-related signaling, we tested the activity of extrinsic and intrinsic apoptosis-related factors by flow cytometry. As illustrated in Figure 2A and B, both Fas and Fas-L were triggered by hyperforin treatment on HT29 and HCT116 cells in a dose-dependent manner. In Figure 2C, the cleavage form of caspase-8 was found to be accumulated by hyperforin treatment in both HT29 and HCT116 cells. Next, we illustrated that hyperforin may disrupt the mitochondria membrane potential (Δψm) which is a major phenomenon of intrinsic apoptosis. As shown in Figure 2D, loss of Δψm was increased by hyperforin. In the meantime, the intrinsic apoptosis-associated factor, cleaved caspase-9, was also shown to be activated in hyperforin-treated cells (Figure 2E). Finally, we performed western blotting to identify whether the protein expression of the above-mentioned factors was affected. Results in Figure 2F show that activation of cleaved caspase-3, -8, -9 and BAK by hyperforin are between 2- to 7-times higher compared to those of the untreated group. In summary, hyperforin initiates an apoptosis effect in CRC cells both through extrinsic and intrinsic apoptosis pathways.

Figure 2. Induction of extrinsic and intrinsic apoptosis by hyperforin on HT29 and HCT116 cells. HT29 and HCT116 cells are treated with different concentrations of hyperforin, and results were evaluated with (A) Fas staining assay, (B) Fas-L staining analysis, (C) cleaved caspase-8 staining assay, (D) DIOC6 staining assay and (E) cleaved caspase-9 staining assay, and (F) Western blotting assay. (*p<0.05, **p<0.01, ***p<0.005 vs. 0 μM hyperforin).

Figure 2

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Hyperforin may effectively suppress the invasion and migration ability of CRC cells. We further investigated whether hyperforin affects the invasion and migration capacity of CRC cells. Hyperforin inhibition of the invasive and migratory properties of CRC cells was proven by the transwell system, as shown in Figure 3A and B. Hyperforin suppressed more than 50% of invasion and migration ability under 5 μM concentration for 24 h. Additionally, the wound healing results also illustrated that the migratory effects of HT29 and HCT116 cells were decreased by hyperforin in a time-dependent manner (Figure 3C). The area of the wound was markedly decreased in the untreated group compared to the 5 or 10 μM hyperforin-treated groups. Finally, we elucidated that the protein expression of anti-apoptosis, proliferation, and metastasis-related factors, such as MCL-1, XIAP, cyclinD1 and MMP9 were all reduced by hyperforin (Figure 3D). All in all, the progression of HT29 and HCT116 cells were markedly suppressed by hyperforin.

Figure 3. Inhibition of invasion and migration ability by hyperforin on HT29 and HCT116 cells. HT29 and HCT116 cells were treated with different concentrations of hyperforin and evaluation of these effects was performed with (A) transwell invasion assay, (B) transwell migration assay, (C) wound healing assay, and (D) western blotting assay (*p<0.05, **p<0.01, ***p<0.005 vs. 0 μM hyperforin; $$p<0.01, $$$p<0.005 vs. 5 μM hyperforin).

Figure 3

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Hyperforin-initiated anti-CRC effect is associated with the inactivation of multi-oncokinase-mediated signaling. After confirming apoptosis induction and metastasis inhibition of hyperforin on CRC cells, we aimed to identify the underlying mechanisms. Thus, we performed a western blotting assay to investigate the alteration of hyperforin on multi-oncokinase, such as JAK1, STAT3, ERK, AKT and their downstream factors NF-ĸB and PRAS40. As indicated in Figure 4A, phosphorylation of JAK1, STAT3 and NF-ĸB was decreased by hyperforin in dose-dependent manner. In addition, phosphorylation of ERK, AKT and PRAS40 was also found to be suppressed by hyperforin (Figure 4B). To conclude, the anti-CRC effect of hyperforin is associated with the inactivation of multiple oncogenic kinases.

Figure 4. Inhibition of multiple oncogenic kinases by hyperforin on HT29 and HCT116 cells. HT29 and HCT116 cells were treated with different concentrations of hyperforin, and quantitation of affected proteins was performed with (A-B) western blotting assay.

Figure 4

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Discussion

Epidermal growth factor receptor (EGFR) signaling initiates phosphoinositide 3-kinases (PI3K)/AKT and RAF/mitogen-activated protein/ERK kinase (6)/ERK pathways to mediate tumor progression. Both ERK and AKT, crucial components of oncogenic signal pathways, control tumor growth, survival, and invasion through triggering activation of downstream signaling cascades (26,27). High expression of phosphor-ERK and phosphor-AKT is associated with poor outcomes in patients receiving irinotecan-cetuximab (28). Herein, we found that the protein levels of both ERK (Thr202/Tyr204) and AKT (Ser473) were reduced by hyperforin treatment of HT29 and HCT116 cells (Figure 4B). PRAS40 is a 40-kDa proline-rich AKT substrate and its phosphorylation participates in tumor growth (29). The results showed that hyperforin also suppressed p-PRAS40 expression (Figure 4B).

Nuclear factor-kappaB (NF-ĸB) is an oncogenic transcription factor composed of p50 and p65 subunits, its activation is implicated in EGFR-mediated tumor progression (30,31). The JAK-STAT3 signaling pathway can be activated by different upstream signaling events such as interleukin-6 (IL-6) and EGFR signaling (32,33). Constitutive activation of both NF-ĸB and STAT3 up-regulates the expression of downstream effector proteins including XIAP, MCL-1, CyclinD-1, and MMP-9 leading to initiation of multiple tumor progression processes such as anti-apoptosis, proliferation, and invasion (3,34,35). Positive expression of NF-ĸB, STAT3, and their downstream effector proteins significantly correlate with metastasis and worse survival in patients with CRC (36-39). Hyperforin was demonstrated to inhibit NF-ĸB activity and invasion-associated proteins encoded by NF-ĸB-related genes leading to inhibition of bladder cancer cell invasion (40). Our data indicated that hyperforin reduced not only NF-ĸB and STAT3 activity, but also the expression of downstream effector proteins (Figure 4A). In addition, the invasion ability of CRC cells was significantly inhibited by the treatment with hyperforin (Figure 3).

Anticancer drugs and radiation induce apoptosis by initiating extrinsic and intrinsic pathways. Both caspase-8 and caspase-9 are initiator caspases which activate executioner caspases to mediate apoptosis in extrinsic and intrinsic pathways, respectively (41). Hyperforin was indicated to effectively induce apoptosis through extrinsic and intrinsic pathways in hepatocellular carcinoma and bladder cancer cells (19,40). In addition to the suppression of anti-apoptotic proteins, we also found that hyperforin induced expression of pro-apoptotic protein BAK and cleavage of caspase-3, -8, and -9 in CRC cells (Figure 1 and Figure 2). According to these data, it is suggested that hyperforin as a potential apoptosis inducer not only initiates apoptotic signaling pathways but also eliminates anti-apoptotic proteins.

In conclusion, hyperforin was demonstrated to possess anti-CRC properties including induction of apoptosis, inhibition of anti-apoptotic, and invasion ability. Suppression of ERK, AKT, NF-ĸB, and STAT3 signaling may be associated with hyperforin-inhibited survival and invasion of CRC cells.

Conflicts of Interest

The Authors declare no competing financial interests regarding this study.

Authors’ Contributions

LCH, FTH, and HFC performed the experiments, derived the models, and analyzed the data. FTH, CYK and JJO prepared the initial version of the paper. FTH, HFC, CYK and JJO conceived of the presented idea, supervised the findings of this work, performed the literature review, and prepared the final versions of the paper.

Acknowledgements

The Authors thank the Medical Research Core Facilities Center, Office of Research & Development at China Medical University (Taichung, Taiwan, R.O.C.) for the technical support.

Funding

This study was supported by National Yang Ming Chiao Tung University Hospital, Taipei, Taiwan, ROC (ID: RD2022-010, RD2022-015) and Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan (ID: BRD-109023), respectively.

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