Synergistic anti-hepatoma effect of triptolide and quercetin via co-inhibition and interaction with Janus kinase and mammalian target of rapamycin signal pathway

Abstract

BACKGROUND

Liver carcinoma, as a major global health concern due to its high incidence and mortality rates. Despite advancements in diagnostic and treatment methodologies, outcomes for hepatocellular carcinoma remain unsatisfactory. In response to these limitations, patients increasingly turn to alternative therapies such as traditional Chinese medicine, which has demonstrated potential in enhancing quality of life and prolonging survival in combination with conventional treatments. Triptolide (TP) and quercetin, as two broad-spectrum antitumor activities, act through multiple mechanisms, and whether the combination of them can provide a synergistic effect to improve the treatment effect. To optimize the dosage combination of TP and quercetin to maximize their therapeutic benefits in treating liver cancer, potentially advancing the field of drug combination therapy. This approach seeks to explore new treatment strategies and elucidate the underlying mechanisms that could lead to improved outcomes for hepatocellular carcinoma patients facing limited effective treatment options.

AIM

To investigate the synergistic anti-hepatoma effect of TP and quercetin and elucidate the underlying molecular mechanism involving the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) and mammalian target of rapamycin (mTOR) signaling pathways.

METHODS

The study utilized 5-week-old female BALB/c-nu mice for establishing a liver cancer subcutaneous transplant tumor model. TP and quercetin were administered intraperitoneally over 21 days to evaluate the effectiveness of the combination, with monitoring of tumor growth. IncuCyte Zoom and CompuSyn software were employed to analyze drug effects for different dose combination on cell proliferation and synergy. Various assays such as CCK-8 cell proliferation analysis, plate cell clone formation, cell scratch experiments, Transwell migration and invasion assays, Annexin V-FITC flow cytometry, and western blotting using specific antibodies were employed to assess cell apoptosis, migration, invasion. Then transcriptome analysis was used RNA sequencing to find the potential synergistic mechanisms and proved by western blotting.

RESULTS

In vivo, the combination therapy significantly slowed down tumor growth compared to the control group, quercetin alone group, and TP alone group. The tumor inhibition rates were 28.91% (quercetin), 28.8% (TP), and 59.3% (combination therapy), respectively. The determination of IncuCyte Zoom and CCK-8 confirmed that there is a concentration gradient and time gradient effect on tumor inhibition, with the synergistic effect of 25 nmol/L TP and 100 μmol/L quercetin being the best. Platelet cell clone formation and cell wound scratch assay showed that the combination group had better inhibitory effects. Transwell analysis showed a decrease in migration and invasion in the combination therapy group. Flow cytometry showed that over time, cell apoptosis increased after combination therapy. Transcriptome analysis emphasizes unique pathways influenced by the combination (JAK-STAT and mTOR signaling pathways) and has been validated at the protein level.

CONCLUSION

Compared with a single drug, the specific metering combination of TP and quercetin has enhanced anti-tumor effects, mediated by inhibition of cell proliferation, inducing cell apoptosis and inhibiting migration/invasion. This synergistic effect is closely related to the simultaneous inhibition of signaling pathways JAK-STAT and mTOR concurrently.

Key Words: Triptolide; Quercetin; Synergistic effect; Janus kinase signal pathway; Mammalian target of rapamycin signal pathway; Liver carcinoma

Core Tip: This study demonstrates that a specific combination of triptolide (25 nmol/L) and quercetin (100 μmol/L) exerts a synergistic antitumor effect against hepatocellular carcinoma both in vitro and in vivo. The combination significantly inhibits tumor cell proliferation, induces apoptosis, and suppresses migration and invasion more effectively than either drug alone. Mechanistically, this synergy is achieved through the simultaneous inhibition of the Janus kinase-signal transducer and activator of transcription and mammalian target of rapamycin signaling pathways, as confirmed by transcriptomic analysis, molecular docking, and western blot validation. Importantly, the combination reduces the required dose of triptolide, thereby mitigating its toxic side effects while maintaining potent anticancer efficacy. This research highlights a promising therapeutic strategy for hepatocellular carcinoma that leverages the complementary actions of natural compounds to enhance treatment outcomes.

INTRODUCTION

Liver carcinoma is one of the malignant tumors with very high morbidity and mortality in the world, which seriously threatens the human health. According to the World Health Organization, there are about 906000 new cases and nearly 830000 deaths each year, which lead to it as the sixth most incidence rate cancer and the third leading cause of cancer death in the world[1]. China accounts for 53% of liver cancer deaths worldwide, and in all of this liver carcinoma, more than 90% are hepatocellular carcinoma (HCC)[2]. Studies have shown that liver cancer is related to long-term chronic hepatitis, which is a long-term inflammatory process, in which inflammatory cells infiltrate liver parenchyma and lead to liver cell death, and then stimulate liver cell proliferation and regeneration[3], and then liver structure destruction leads to cirrhosis, liver cell degeneration and formation of dysplastic nodules, which is significantly related to the risk of liver cancer. Eventually, malignant lesions occur[4].

Although with the popularization of testing and the development of the treatment method, the treatment of liver carcinoma is still extremely difficult because it is usually already advanced when the disease is detected, and the treatment effect is far from reaching the expectations[5]. The continuous development of new technologies and drugs provides hope for further progress, including immunotherapy and targeted therapy, which have also improved the survival rate of liver cancer patients[6,7]. However, the age standardized 5-year relative survival rate of HCC is still only 18.1%[8]. Two clinically approved targeted therapy drugs, sorafenib and lenvatinib, can only prolong the overall survival by 2-3 months[9,10]. At present, although multiple comprehensive treatments for liver cancer have achieved certain success, they still faces challenges such as low objective response rate and adverse treatment reactions. Therefore, comprehensive analysis should be conducted from multiple aspects to explore more effective treatment methods and adopt a combination therapy strategy, in order to further improve the treatment effect of HCC.

Because of the poor prognosis and limited treatment options, patients may seek alternative treatments, including traditional Chinese medicine (TCM). TCM is an important component of comprehensive cancer treatment that improves quality of life, delays cancer progression, and prolongs median survival time for patients[11]. Clinical studies have shown that the combination of TCM with interventional therapy can improve the overall effective rate and delivery time of primary HCC patients[12,13]. In basic research, TCM formulas and various their monomers have a positive effect on the occurrence, proliferation, invasion, metastasis, and angiogenesis of liver cancer[11,14]. Like traditional chemotherapy methods, there are often multiple chemical reagents combined. This study also attempts to explore effective combination methods of TCM monomers, especially their synergistic effects, in order to further improve treatment efficacy.

Triptolide (TP), a bioactive diterpenoid from Tripterygium wilfordii Hook F, exhibits potent anticancer effects across solid tumors, including HCC, by attenuating transcriptional outputs [e.g., signal transducer and activator of transcription 3 (STAT3)-dependent survival genes], disrupting chaperone/proteostasis functions, and ultimately curbing proliferation and invasion[15-17]. Quercetin is known for its anticancer potential and has been shown to exert its chemopreventive and anticancer effects through various pathways, such as antioxidant and anti-proliferative effects, cell cycle arrest, apoptosis induction, and inhibition of cell invasion and angiogenesis[18-22]. Both TP and quercetin have shown great potential to inhibit or kill tumors. In parallel, TP has been linked to modulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) readouts and apoptosis induction, although it exhibits significant toxic side effects, mainly manifested as drug-induced liver and kidney injury, which necessitates rational combinations and dose-sparing regimens[23,24]. Critically, quercetin has been extensively studied for its hepatoprotective properties, with evidence showing its ability to mitigate TP-induced liver injury by modulating inflammatory and immune pathways[25]. Therefore, we will combine these two drugs to explore the optimal dosage combination for their synergistic effect in the treatment of liver cancer, in order to explore the potential of drug combination therapy and potential mechanisms of this action.

MATERIALS AND METHODS

Materials and chemicals

Corning RPMI 1640 culture medium (10-040-CV) was used for cell culture. The standard substances of TP (CAS No. 38748-32-2, MUST-19111310, 20 mg) and quercetin (CAS No. 117-39-5, MUST-19101104, 20 mg) was purchased from Chengdu Mansit Biotechnology Co., Ltd (China). Annexin V-FITC flow cytometry test kit (CAS No. 559763, BD Company, NJ, United States) was used to detect apoptosis by flow cytometry. 8 μm 24 well plate Transwell chamber (CAS No. 3422, Corning, NY, United States) was used to detect the migration and invasion. The antibody of Bax (CAS No. 5023, Cell Signaling, MA, United States), Bcl-2 (CAS No. 3498, Cell Signaling, MA, United States), P53 (CAS No. 2527, Cell Signaling, MA, United States), caspase-3 (CAS No. 14220, Cell Signaling, MA, United States), cleaved caspase-3 (CAS No. 9978, Cell Signaling, MA, United States), Janus kinase 1 (JAK1) (CAS No. Ab125051, Abcam, United Kingdom), p-STAT3 (CAS No. Ab267373, Abcam, United Kingdom), PI3K (CAS No. Ab191606, Abcam, United Kingdom), p-PI3K (CAS No. Ab278545, Abcam, United Kingdom), AKT (CAS No. Ab179463, Abcam, United Kingdom), p-AKT (CAS No. Ab8805, Abcam, United Kingdom), mTOR (CAS No. Ab2732, Abcam, United Kingdom), p-mTOR (CAS No. Ab109268, Abcam, United Kingdom) was used to detect the expression level of related proteins.

Animal and cell

The HepG2 was purchased from China Center for Type Culture Collection. 24 5-week-female BALB/c-nu mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd (animal qualification certificate number: 11400700263331. production license number for experimental animals: SYXK2011-0024). All of BALB/c-nu mice were raised in pre-disinfected IVC cages at a temperature of 22-26 °C, a humidity of 40%-60% under a 12-hours dark/Light cycle with standard food and clean water in a specific pathogen-free barrier environment. All of BALB/c-nu mice were given one week to adapt to the new environment. Animal ethics was permitted by the Institute of Basic Theory for Chinese Medicine, China Academy of Chinese Medical Sciences.

Construction and intervention of BALB/c-nu nude mouse liver cancer subcutaneous transplant tumor model

When HepG2 cells grow into logarithmic growth phase, digest the cells to obtain cell resuspension, and obtain a final cell concentration of 4.33 × 10⁶/mL. Disinfect the axillary skin of BALB/c-nu female nude mice with iodine, mix the cells from the previous step, and use a 1 mL sterile syringe to aspirate and resuspend 100 μL (4.33 × 10⁵/per mouse), with the needle at an angle of about 20° to the skin, gently lift the skin from the side ribs, insert the needle along the armpit, and then inoculate the cell resuspension subcutaneously in the armpit. Around 10 days later, the tumor can form with a diameter of about 5 mm, indicating successful implantation of the tumor. Number the mice properly, measure long and short diameters of the tumor and record them. Calculate the approximate volume (V) of the tumor according to the formula: V = 1/2 (length × width²)[26]. Arrange the tumors according to their volume and divide them into four groups using a stratified random sampling method using a random number table to ensure that the tumor volume of each cage of mice is relatively uniform. After 10 days of tumor formation in nude mice, each group will receive a 21 days intervention, and drug injection will be administered intraperitoneally. Here, quercetin was used 40 mg/kg/day per mouse (intraperitoneal injection)[27-29], TP was used 500 μg/kg/day per mouse (intraperitoneal injection)[30-32]. Give the quercetin 20 minutes after administration, and then administer the TP[25,33,34]. The detail administration for the intervention style and the grouping were in the Supplementary Table 1. In the continuous 21-day intervention, we would record the nude mice’ state for each 7 days contained the length and width of tumor, weight, food-intake and so on. After 21 days’ administration, nude mice were euthanized using cervical dislocation method, and the cortex on the tumor was carefully peeled off to observe the growth status of the tumor and weighed; simultaneously, liver samples were taken and further routine pathological sections were performed. Considering the first drug dissolved in DMSO, we maintained the same concentration of DMSO in the final administration and adopted a two-doses approach. In addition, we also took liver and did hematoxylin and eosin staining to evaluate the hepatotoxicity of the drug.

The different dose combinations effect in anti-tumor effect and data analysis by IncuCyte Zoom

IncuCyte ZOOM (Essen Bioscience, MI, United States) is a non-invasive, long-term real-time dynamic live cell imaging analysis platform. The signal acquisition machine can be placed in a culture incubator, with various culture containers placed in the middle. There is a micrograph device below it, which continuously monitors the cultured cells through micrographs and can be remotely controlled, data read and analyzed. 96 well plate for HepG2 tumor cell seeding and drug addition, with 2500 cells per well. Place the stationary 96 well plate in IncuCyte Zoom in the incubator and select 10 × phase contrast objective, set to capture 4 images per hole, and cycle the shooting mode every 3 hours. Use the system’s built-in analysis software (IncuCyteZOOM 2015A, Essen Bioscience, MI, United States) to collect information for 48 hours. This experiment should be repeated at least twice. Open the captured image window and analyze it in the Analysis Job Utilities displayed in the upper right corner of the software. After completion, various data such as cell saturation, number, etc. can be viewed in the table below the image. The detail dose combination of TP and quercetin see Supplementary Table 2.

Calculate the synergistic or antagonistic effects of drugs under different dosage combinations by CompuSyn (Chou Talalay method)

Chou[35] used Chou Talalay method and developed the software “CompuSyn” in the analysis of the third-generation drug combination action dose-response, further improve the theory of this method and summarize its research in combination therapy[36]. The Chou Talalay method can provide a combination index, which not only reflects the additive effect, synergistic effect, and antagonistic effect of combination therapy, but also provides a description of dose-response level effects. The final results will be represented by inhibition rate combination index and equivalent line graph (isobologram), respectively. Calculate the cell activity obtained from IncuCyte Zoom using CompuSyn1.0 software and generate the combined effects of two drugs at different doses. The cell proliferation inhibition rate of each group was calculated according to IncuCyteZOOM 2015A, and the combined effect results were calculated after data of each group were input respectively.

CCK-8 cell viability assay

Take logarithmic growth phase cells, grow 2500 cells per well on a 96 well plate, and pre culture in a culture incubator for 24 hours (at 37 °C, 5% CO₂ conditions). Add different concentrations of drugs according to experimental requirements. They are blank control group, TP 10 nmol/L, TP 25 nmol/L, TP 50 nmol/L, quercetin 75 μmol/L, quercetin 100 μmol/L, quercetin 150 μmol/L, TP 25 nmol/L + quercetin 75 μmol/L, and TP 25 nmol/L + quercetin 100 μmol/L, respectively. Incubate the culture plate containing the medicine in the incubator for an appropriate period of time (24 hours, 48 hours, 72 hours); considering that the reducing performance of quercetin reacts with CCK-8, a plate centrifuge of 1000 rpm for 5 minutes is required before adding CCK-8. The supernatant is removed and gently washed once with phosphate buffered saline (PBS). The mixture is added to 96 wells at a ratio of 10 μL of CCK-8 100 μL/well of culture medium, incubated for 2 hours, and the 450 nm absorbance is measured.

Plate cell clone formation test

Take logarithmic growth phase HepG2 cells, digest and blow them into individual cells, and resuspend the cells in the culture medium; dilute the cell suspension step by step according to the gradient to 500 cells/mL, add 2 mL per well on a 6-well plate totally 1000 cells per well, and blow the cells evenly. Incubate in a 37 °C 5% CO₂ constant temperature cell culture incubator for 24 hours; after 24 hours, add the drugs TP 25 nmol/L and quercetin 100 μmol/L, TP 25 nmol/L+ quercetin 100 μmol/L, blank control group [RPMI 1640 containing 0.1% DMSO and 10% foetal bovine serum (FBS)], incubated in a constant temperature cell culture incubator for 24 hours and 48 hours; after 24 hours and 48 hours (two time points), the drug was aspirated and discarded separately (the control group was the culture medium). The 6-well plate was rinsed with PBS twice to remove residual drug, and fresh RPMI 1640 containing 10% FBS (pre water bath to 37 °C) was replaced for continuous cultivation for 10 days. Finally, the number of clones was determined by crystal violet staining. Clone formation rate = (number of clones/number of inoculated cells) × 100%.

Wound healing assay

Logarithmic growth phase HepG2 tumor cells were added with 2 mL of cell resuspension (5 × 10⁵ cells/well) into 6 plate well. After 24 hours, a 10 μL pipette was used to scratch along the straight edge as much as possible along the previous posterior horizontal line. Wash the cells with PBS three times, and remove the scratched cells and cell debris. Add 25 nmol/L TP and 100 μmol/L quercetin, TP 25 nmol/L + quercetin 100 μmol/L, blank control group (all used medium contained 0.1% DMSO without FBS). Take photos and record them for 0 hour, 12 hours, and 24 hours respectively; calculate the displacement distance and area percentage during the cell scratch experiment using ImageJ.

Transwell assay

Before preparing the cell suspension, the cells were starved without FBS for 24 hours to further remove the influence of FBS. Resuspend logarithmic growth phase HepG2 tumor cells in 1% FBS medium. Each upper chamber of 24 well plate would contain 5 × 10⁴ cells. Add 600 μL of culture medium containing 10% FBS or prepared drugs with 10% FBS to the 24 well plate lower chamber. As to the upper chamber, add drugs containing 1% FBS or culture medium containing 1% FBS to the upper chamber, then add 5 × 10⁴ cells. After adding, gently blow the cells evenly. The order of drug addition for Transwell detection of cell migration and invasion was in Supplementary Table 3. After 12 hours culture, remove the medium and wash twice with PBS. Fix a new 24 well plate with 600 μL of 4% paraformaldehyde for 30 minutes and wash with distilled water for 2 minutes. 24 well plate with 600 μL 0.1% crystal violet staining for 20 minutes, gently rinse the lower layer of the upper chamber with a pipette, and then rinse three times with PBS. Observe cell counts in five random fields under a 200 × microscope.

For the invasion experiment, matrigel was thawed in a 4 °C environment, diluting 5 mL of matrix adhesive at a ratio of 1/40, and finally diluting RPMI 1640 on ice to form a solution containing 1% FBS and 2.5% matrix adhesive. Add 100 μL of Transwell to each upper chamber, ensured that there are no bubbles, and then placing it in a 37 °C constant temperature incubator for 2-3 hours until it solidifies. Finally, absorb and precipitate the liquid. Then add drug and cells according to the migration experiment above.

Detection of cell apoptosis using Annexin V-FITC/PI dual staining flow cytometry

After inoculating HepG2 tumor cells with logarithmic growth phase into R-6 culture dish, add the drugs TP 25 nmol/L and quercetin 100 μmol/L, TP 25 nmol/L + quercetin 100 μmol/L, blank control group (RPMI 1640 with normal 10% FBS), incubated in a constant temperature cell culture incubator for 24 hours and 48 hours. After digestion with pancreatic enzyme, transfer to a 2 mL centrifuge tube, centrifuge 400 × g for 5 minutes, at 4 °C (pre cooled). After centrifugation, take out a 2 mL centrifuge tube, discard the supernatant, add 1 mL PBS, blow well and disperse the cells, and transfer the PBS containing cells to a 1.5 mL centrifuge tube. Repeat the centrifugation process once. Add 5 μL FITC + 5 μL PI to each experimental group, control 4 wells (PI + FITC, FITC, PI, none), and incubate at room temperature in dark for 10 minutes. Blow the cells evenly and resuspend them, filter them through a filter membrane and transfer them to a flow cytometer. Test the cell apoptosis rate on the machine. Analyze cell apoptosis using FlowJo 10 and calculate the percentage of various types of cells.

Transcriptome analysis and comparison of synergistic drug efficacy

Logarithmic growth phase HepG2 tumor cells were cultured in 6-well plate with the addition of drugs TP 25 nmol/L and quercetin 100 μmol/L, TP 25 nmol/L + quercetin 100 μmol/L, blank control group (RPMI 1640 with normal 10% FBS), respectively. The drug has a duration of action of 12 hours, with three reconstructions performed for each treatment. RNA was extracted using QIAGEN RNeasy Mini Kit (Cat No./ID: 74104, Germany). The total RNA concentration, RIN value, 28S/18S, and fragment size were detected using Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit, China). A library was established and sequenced on the machine. Perform differential expression gene pathway functional analysis through the website (https://www.bioinformatics.com.cn/). By comparing the blank control group, we explore new pathways emerging in combination therapy by comparing single drugs.

Molecular docking

In this study, ligand molecules, including TP and quercetin obtained from the PubChem database (PubChem CID: 107985 and 5280343), were saved as sdf files. These files were converted into pdbqt format using Open Babel software. The three-dimensional structures of JAK1, STAT3, PI3K, and mTOR proteins (PDB ID: 6GGH, 6NJS, 3APC, 6ZWO) were retrieved from the PDB protein database. These protein structures were imported into AutoDock Tools and saved in pdbqt format. Docking simulations were performed using AutoDock Vina with default docking center positions and grid sizes covering the entire protein structure. Binding energies were assessed based on the docking poses and interactions between the ligand molecules and the receptors.

Western blot

HepG2 cells were inoculated in the 6-well plate and intervened when the cell density reached 70%-80%. Cells were inoculated in the six-well plate as required (TP 25 nmol/L, quercetin 100 μmol/L, TP 25 nmol/L+ quercetin 100 μmol/L, blank control group), and treated for 48 hours. After treatment, the medium was sucked out, each well was washed thrice with precooled PBS, and 100 μL RIAP lysate buffer was added to treat cells for 10 minutes on the ice, centrifuged at 12000 rpm for 15 minutes, and the supernatant was collected and quantified by the BCA method. Sodium-dodecyl sulfate gel electrophoresis gel was prepared, after the separation gel was taken out and put into the electrophoresis solution. The polyvinylidene fluoride (PVDF) membrane was activated in methanol solution for 10 minutes and then equilibrated in electrophoresis solution. The adhesive and membrane were carefully bonded together and put into the membrane transfer tank for membrane transfer. After the membrane was transferred, it was sealed in 5% skimmed milk powder for 1 hour and incubated overnight at 4 °C. The next day, TBST was used to wash the PVDF membrane 3-5 times for 5 minutes each time. The second antibody was added and incubated at room temperature for 1 hour. Then, the PVDF membrane was washed by TBST 3-5 times; add the substrate developer and take color photos in the dark room.

Statistical analysis

The experimental data was statistically analyzed using SPSS 20.0 software. Measurement data are expressed as mean ± SD. Multigroup econometric data were analyzed using one-way ANOVA. A P value less than 0.05 was considered to be statistically significant.

RESULTS

Inhibitory effect of TP combined with quercetin on transplanted tumor in nude mice

After 3 weeks of intervention, tumor volume growth in the combined treatment group was significantly slower than that in the other three groups, with statistical differences compared with the control group, quercetin group and TP group (Figure 1A-C). In terms of tumor weight, quercetin group and TP group had no statistical difference with the control group as reference, but the P value approached 0.05, while there was a statistical difference compared with the combined treatment group, suggesting that the combined treatment could significantly slow down the tumor growth. According to the average tumor weight in each group, tumor inhibition rate was found to be 28.91% in the quercetin group, 28.8% in the TP group, and 59.3% in the combined drug group, suggesting that combined drug use can achieve better tumor inhibition effect (Figure 1D and E). Thus, compared with TP group and quercetin group, the combination of drugs showed a stronger antitumor effect.

Figure 1 The anti-tumor effect of triptolide and/or quercetin in vivo. A: The tumor of the nude mice after 21 days’ treatment; B: All tumors stripped from nude mice in the four groups; C: The change of the mean volume of tumor in each group; D: The mean weight in each group after 21 days’ treatment; E: The tumor inhibition rate compared with control group; F: The mean loss of weight in each group after 21 days’ treatment; G: The change of the mean weight of tumor in each group; H: The change of the mean weight of food-intake in each group. TP: Triptolide; QU: Quercetin.

As the intervention progressed, the TP group experienced the greatest weight loss, followed by the combination group, while the control group experienced the slowest weight loss (Figure 1F and G). The weight loss in the combined treatment group was lower than that in the TP group, with statistical difference, suggesting that quercetin may be helpful in protecting the body from TP toxicity. The weight loss in control group and quercetin group was also significantly lower than that in TP group, with a statistically significant difference. In terms of food intake, there was an interesting performance, in the early stage of TP administration (the first week), the food intake of this group was higher than that of other groups, but with the progress of intervention, the food intake decreased rapidly, and then reached a similar level with that of other groups at the end. However, the diet of the other groups was relatively stable and at a similar level (Figure 1H). Pathological evaluation of long-term drug-induced liver damage see Supplementary Figure 1.

IncuCyte ZOOM was used to screen the anti-tumor effects of different dose combinations of TP and quercetin

After 48 hours of drug action, different dose combinations had different effects on the proliferation curve of HepG2 tumor cells (Figure 2, detail dose combinations see Supplementary Table 2). Totally, for the single drug, it showed that it is a typical relationship of concentration dependent effect and time dependent effect. When TP 25 nmol/L combined with quercetin 100 μmol/L, its inhibitory effect on tumor proliferation was similar to that of higher dose of TP (> 25 nmol/L) combined with quercetin, and it could effectively inhibit the proliferation of HepG2 cells (Figure 3 and Supplementary Figure 2). While, when TP 25 nmol/L combined with quercetin whose dose lower than 100 μmol/L, the proliferation inhibition began to weaken rapidly (Figures 2 and 3, Supplementary Figure 3). When the concentration of TP is 12.5 nmol/L and 5 nmol/L, the combinational inhibitory effect with arbitrary dose of quercetin on tumor cell proliferation was much lower than that of 25 nmol/L TP combined with the corresponding dose of quercetin (Figure 2).

Figure 2 The proliferation curves of different dose combinations between quercetin and triptolide using IncuCyteZOOM 2015A. The combination of various doses was added to the cells and they were cultured in IncuCyteZOOM 2015A. We took pictures of each group every 3 hours. Then the software could change them into data of proliferation. TP: Triptolide; QU: Quercetin.

Figure 3 The combination of 25 nmol/L triptolide with 100 μmol/L quercetin. A: The sequence pictures of the combination of 100 μmol/L quercetin and 25 nmol/L triptolide in 48 hours in the same point by live cell workstation (IncuCyteZOOM 2015A); B: The proliferation curve for 25 nmol/L triptolide combined with 100 μmol/L quercetin in 48 hours; C: The combination index for the combination of 25 nmol/L triptolide with 100 μmol/L quercetin calculated by CompuSyn. TP: Triptolide; QU: Quercetin.

CompuSyn method was used to calculate the combined effects of drug combinations in each group

After obtaining the growth rate through IncuCyteZOOM 2015A, the inhibition rate of each group was calculated. After the inhibition rate of each group was brought into CompuSyn1.0, the joint application index of each combination could be output (Table 1 and Supplementary Figure 4). Among all of these dose combinations, TP 25 nmol/L combined with quercetin 100 μmol/L showed the best combined effect, with a combined index of 0.31205. From IncuCyteZOOM 2015A’s recording of temporal images, under this dose combination, the cells showed obvious cell morphological changes, the cells shrank and became round, the antennae disappeared, and the intercellular connections disappeared (Figure 3). Under the coordination of quercetin and the application of low dose of TP (25 nmol/L), the effect of anti-tumor was still very good.

Table 1 The combination index calculated by CompuSyn.

	TP (100 nml/L)	TP (75 nml/L)	TP (50 nml/L)	TP (25 nml/L)	TP (12.5 nml/L)	TP (5 nml/L)
QU (100 μmol/L)	0.70667	0.55290	0.39527	0.31205	0.63441	0.73873
QU (75 μmol/L)	0.81212	0.56446	0.38721	0.51251	0.98118	0.79892
QU (50 μmol/L)	0.76843	0.57304	0.42373	0.91086	1.17449	1.29329
QU (25 μmol/L)	1.07469	0.87244	0.64508	1.51069	1.21905	1.42084
QU (12.5 μmol/L)	1.04648	0.73518	0.72630	1.31242	1.51697	1.58935

TP: Triptolide; QU: Quercetin.

Result of CCK-8 cell viability assay

Totally, for these two single drugs, they showed the similar effect compared to IncuCyteZOOM analysis, that the anti-tumor effect of TP and quercetin presented time dependency and dose dependency. It showed lower cell viability when 25 nmol/L TP combined with 100 μmol/L quercetin, compared with 25 nmol/L TP or 100 μmol/L quercetin alone, similar to the effect of 50 nmol/L TP (Figure 4A). At the same time, the effect of 25 nmol/L TP combined with 100 μmol/L quercetin was gradually enhanced with the extension of time. The inhibitory rate of 25 nmol/L TP combined with 75 μmol/L quercetin was slightly lower at all time points than that of 25 nmol/L TP combined with 100 μmol/L quercetin. The inhibitory effect of TP combined with quercetin was increased when the dosage of any one of these two drugs was elevated. At 72 hours, the cell activity or cell growth inhibition rate of TP 25 nmol/L combined with quercetin 100 μmol/L was statistically significant with other groups (all P values < 0.05) except TP 50 nmol/L group (P = 0.65) (Figure 4B and C).

Figure 4 Combinational anti-tumor effect of triptolide and quercetin in HepG2 cell line compared with single drug tested by CCK-8. HepG2 cell cells were treated with 25 nmol/L triptolide alone or combined with 75 μmol/L or 100 μmol/L quercetin for 24 hours, 48 hours, 72 hours treatment. A: The OD value determined by CCK-8 assay in 24 hours, 48 hours, 72 hours treatment; B: Cell viability ratio in each group; C: Cell growth inhibition ratio in each group. The tests were performed at least three independent parallel experiments. All of the data were expressed by mean ± SD. ^aP < 0.05 compared to other groups excluded triptolide 50 nmol/L group. TP: Triptolide; QU: Quercetin.

Plate cell clone formation test

After 24 hours or 48 hours of intervention, the number of clone formation in three drug group were lower than control group. Meanwhile, the inhibition of clone formation for combinational group is significantly lower than other three group, reflecting a good joint effect. As to the single drug, they all showed effective inhibition of clone formation compared with control group after 24 hours or 48 hours. There was no statistical difference between quercetin and TP after 24 hours of intervention, but there was after 48 hours of intervention (Figure 5).

Figure 5 The colony formation of triptolide and/or quercetin in HepG2 cell line. A: The photos of HepG2 cell line’s colonies with 1000 seeding cell number that treated with 25 nmol/L triptolide and/or 100 μmol/L quercetin for 24 hours; B: The photos of HepG2 cell line’s colonies with 1000 seeding cell number that treated with 25 nmol/L triptolide and/or 100 μmol/L quercetin for 48 hours; C: Quantitative representation of the colonies of HepG2 cells. Compared to the control group, the colony formations in treated groups were decreased and the combinational group was lowest. Meanwhile, the colony formation decreased as time goes on in each drug groups. Data was presented as means ± SE of three independent experiments. ^aP < 0.05. TP: Triptolide; QU: Quercetin.

Effect of combination drugs on wound healing

After 24 hours of drug treatment, the scratch healing of the cells in the blank control group was the best, and the cells at the edge of the scratch were relatively more spread and flatter, and the scratch healing of the TP 25 nmol/L group and the quercetin 100 μmol/L group were worse. TP 25 nmol/L combined with quercetin 100 μmol/L had the lowest moving distance (Figure 6A and B). Draw the straight-line distance by dividing it into eight equal parts using ImageJ, the distance between the combinational group and the other three groups was significantly different (Figure 6B). In terms of single drug, TP 25 nmol/L or quercetin 100 μmol/L also had a statistically significant difference compared with control group. Using ImageJ to calculate the proportion of the scratch area before and after the scratch experiment to the total area (Figure 6C), the scratch area of the blank control group was reduced by 6.74%, the scratch area of quercetin 100 μmol/L was reduced by 5.72%, and the scratch area of TP 25 nmol/L was reduced by 5.45%, and the scratch area of TP 25 nmol/L combined with quercetin 100 μmol/L was reduced by 4.21% after 24 hours, which was generally consistent with the trend calculated by distance.

Figure 6 Combinational effect of triptolide and quercetin in migration tested by wound-healing assay. A: The migrational pictures for four groups after 24 hours treatment; B: Migrational distance for each group after 24 hours tested by ImageJ; C: Area of migration for each group after 24 hours tested by ImageJ. Data was presented as means ± SE. ^aP < 0.05. TP: Triptolide; QU: Quercetin.

Effect of combination drugs on cell migration and invasion

Under the 200-fold field of view, five different fields were randomly selected in each group, and the number of cells migrating to the back of the upper chamber was counted and averaged respectively. After three consecutive experiments, it was found that there was no statistical difference between the blank control group and the single drug group. There were significant differences between the combined drug group and the single drug group and the blank control group (Figure 7A and B).

Figure 7 The migration and invasion of HepG2 tested by Transwell in each group. A: The picture of migration and invasion. The migration and invasion of HepG2 showed best result in the group of 25 nmol/L triptolide combined 100 μmol/L quercetin; B: The numbers of migrational cell. It showed the combinational group had statistically significant difference compared with other groups after 12 hours treatment; C: The numbers of invasional cell. The combinational group had statistically significant difference compared with control group and quercetin group after 24 hours treatment, meanwhile the P value is low compared to triptolide (P = 0.058). The tests were performed at least three independent parallel experiments. Data was presented as means ± SE of three independent experiments. ^aP < 0.05. TP: Triptolide; QU: Quercetin.

As to the cell invasion ability, under the 200-fold field of view, five different fields were randomly selected in each group, and the number of cells invading the back of the upper chamber was counted respectively. It was found that there was no statistical difference between the blank control group and the single drug group. There was statistical difference between the combined treatment group and quercetin group and blank control group, although there was no statistical difference with TP group, the P value was also small (TP + quercetin vs TP P = 0.058, Figure 7A and C).

Effect of combination drugs on apoptosis tested by flow cytometry

By using the Annexin V-FITC/PI dual staining method to detect the apoptosis of cell, we compared the difference in inducing apoptosis between TP and quercetin alone or their combination after 24 hours and 48 hours of intervention. Overall, each group showed a significant increase of apoptosis compared to 24 hours after 48 hours of medication, indicating a time-dependent effect in promoting cell apoptosis. At 24 hours, the apoptosis rate of the combination therapy group was significantly higher than that of the control group and the quercetin group. By 48 hours, the apoptosis rate of the combination therapy group was significantly higher than that of each group (Figure 8). Through flow cytometry, it can be seen that the combination of the two drugs can significantly promote tumor cell apoptosis over time, which is consistent with the gradual decrease in OD value of CCK-8 over time.

Figure 8 Apoptotic effects of triptolide and/or quercetin in HepG2 cell line by fluorescence activated cell sorter analysis. A: Annexin V-FITC/PI was performed to measure the apoptosis and then deal with FlwoJo 10. The pictures showed the treated cells in the four quadrants; B and C: The percentage of apoptotic cells in difference phases in 24 hours or 48 hours. Percentages of negative represent viable cells, annexin V-positive represent early apoptotic cells, PI-positive represent necrotic cells, or annexin V and PI double-positive represent late apoptotic cells. The tests were performed at least three independent parallel experiments. Data was presented as means ± SE of three independent experiments. ^aP < 0.05. TP: Triptolide; QU: Quercetin.

Transcriptome analysis and comparison of synergistic drug efficacy

Compared with the blank control group, we found that there were 10 differential signaling pathways after quercetin treatment, 34 differential signaling pathways after TP treatment, and 31 differential signaling pathways after TP + quercetin treatment (Figure 9A-C showed the top 10 pathways, and Supplementary Table 4 showed all pathways with statistical differences) through transcriptome analysis. Based on this, we intersected the three treatment groups using a Venn diagram and found that TP + quercetin had 8 separate signaling pathways (Figure 9D), namely JAK-STAT signaling pathway, mTOR signaling pathway, glycosphingolipid biosynthesis, maturity onset diabetes of the young, viral life cycle - human immunodeficiency virus type 1, measles, type II diabetes mellitus, legionellosis. Thus, JAK-STAT signaling pathway and mTOR signaling pathway (JAK-STAT/PI3K/AKT/mTOR) may be the main affected pathways for the synergistic effect of quercetin + TP according to previous studies. Similar to previous studies, it has found that the combination of these two drugs (JAK kinase inhibitors and PI3K/mTOR inhibitors) can produce synergistic effects which can benefit cancer patients who are unresponsive or resistant to existing therapies[37].

Figure 9 Pathway functional analysis by Kyoto Encyclopedia of Genes and Genomes compared with control group based on RNA sequencing. A: The top ten signaling pathways with the greatest statistical difference for control group compared with quercetin; B: The top ten signaling pathways with the greatest statistical difference for control group compared with triptolide; C: The top ten signaling pathways with the greatest statistical difference for control group compared with triptolide + quercetin; D: The Venn diagram of signaling pathways for three different drug interventions groups compared with control group. TP: Triptolide; QU: Quercetin.

Molecular docking analysis

The affinity of simultaneous binding of TP and quercetin to the protein targets was evaluated using molecular docking methods. Lower binding affinities indicate stronger binding activity of the compounds to the proteins. As illustrated, JAK1, STAT3, PI3K, and mTOR proteins can simultaneously bind both small molecules without steric clashes, demonstrating favorable docking performance. The quercetin and TP molecules are shown in yellow and blue, respectively (Figure 10). The affinities of quercetin and TP for simultaneous binding to JAK1 were -8.6 kcal/mol and -6.4 kcal/mol, to STAT3 were -8.1 kcal/mol and -7.4 kcal/mol, to PI3K were -7.5 kcal/mol and -7.3 kcal/mol, and to mTOR were -7.7 kcal/mol and -6.8 kcal/mol, respectively. These results further suggest that the co-binding of both molecules may induce different effects compared to single-molecule interactions.

Figure 10  Molecular docking analysis of the two molecules with targets. A: Quercetin and triptolide (TP) docked with Janus kinase 1; B: Quercetin and TP docked with signal transducer and activator of transcription 3; C: Quercetin and TP docked with phosphoinositide 3-kinase; D: Quercetin and TP docked with mammalian target of rapamycin. Jak1: Janus kinase 1; Stat3: Signal transducer and activator of transcription 3; PI3K: Phosphoinositide 3-kinase; mTOR: Mammalian target of rapamycin.

Western blot

We used the drugs TP 25 nmol/L and quercetin 100 μmol/L alone or their combination for 48 hours, and detect the changes in related apoptotic proteins at this time point. The expression of apoptosis inhibitory proteins Bcl-2 and caspase-3 decreased compared to the control group, with the largest decrease observed in the combination therapy group; the ratio of Bax, P53 protein, cleaved caspase-3, and the ratio of Bax/Bcl-2 increased, with the largest increase observed in the combination therapy group (Supplementary Figure 5). The level of JAK1, p-STAT3, mTOR, p-mTOR, p-PI3K, p-AKT was decreased in combination group (quercetin + TP) significantly compared with other three groups, while, there was no statistical difference for the level of PI3K and AKT (Figure 11).

Figure 11  The expression level of related mechanism protein. A: Western blots showing the levels of Janus kinase 1, p-signal transducer and activator of transcription 3, phosphoinositide 3-kinase, p-phosphoinositide 3-kinase, protein kinase B, p-protein kinase B, mammalian target of rapamycin, and p-mammalian target of rapamycin; B: The expression of each protein normalized by β-actin according to grey value. ^aP < 0.05. TP: Triptolide; QU: Quercetin; Jak1: Janus kinase 1; Stat3: Signal transducer and activator of transcription 3; PI3K: Phosphoinositide 3-kinase; AKT: Protein kinase B; mTOR: Mammalian target of rapamycin.

DISCUSSION

At present, although the treatment of liver cancer has achieved some success, it still faces challenges such as low objective remission rate and adverse treatment reactions[38]. Right now, there are many methods to treat liver cancer, but each method seems to have certain limitations. At the same time, most HCC patients are already in the advanced stage of liver cancer at the time of diagnosis, and treatments with potential for healing, such as transplantation, local ablation, and resection, are not appropriate at this stage[5]. So, when liver cancer develops to an advanced stage, its treatment options are limited that lead it still one of the most difficult malignant tumors to treat.

Therefore, in the case that a single treatment mode or single drug cannot meet the treatment needs, it is necessary to combine multiple treatment modes and adopt a combination treatment strategy, so as to seek an effective combination mode, especially a synergistic and efficient joint regimen. However, how to effectively combine is still an urgent problem to be solved. Traditional Chinese herbs contain many natural anti-tumor components, such as flavonoids, quinones, alkaloids and so on. TP is one of the main active components of Tripterygium wilfordii Hook F, which is one highly active epoxidated diterpene lactone compound and has strong anti-infection, anti-immune, anti-tumor and other pharmacological effects[39]. Many studies have shown that TP’s anti-tumor effect is multi-directional, multi-target, cross-play, and broad-spectrum[16,40]. While, its toxic side effects have also received attention, and its shortcomings of narrow dosage range. As to quercetin, it is a well-known flavonoid, and its anticancer properties are related to various cell signaling mechanisms and its ability to inhibit carcinogen-related enzymes, which had been shown inhibition effect in vitro studies, some animal and human studies[41,42]. Studies have shown that quercetin can regulate almost all cancers in a number of ways[43]. Astonishingly, quercetin can reduce the hepatotoxicity for the normal liver tissue caused by TP. Through the interaction of the two anti-liver cancer aspects, the relatively low dose of TP combined with quercetin can still achieve the purpose of tumor inhibition, so as to effectively achieve the aim of enhancing efficiency and reducing toxicity.

First of all, through animal experiments, the initial exploration of the simultaneous administration of two drugs in the treatment of liver cancer transplant tumor model has a good synergistic result, and can reduce side effects, suggesting that the two have a good combination potential. We hope that by exploring the optimal dose combination of the two drug combinations at the cell level, we finally determined that the optimal dose combination was 25 nmmol/L TP and 100 μmol/L quercetin through living cell workstation and CompuSyn method, and the two dose combinations could achieve the maximum synergistic effect in all combinations. At the same time, due to the low concentration of TP, the toxicity of TP to cells is low when acting alone, which can reduce the toxic side effects of TP to some extent and overcome the shortcomings of the narrow safe dosage range of TP. Therefore, we conducted cell activity assay CCK-8, cell cloning assay, cell scratch assay, Transwell experiment detect the migration and invasion, flow detection of apoptosis and other experiments, it was found that this dose combination can effectively inhibit the proliferation of liver cancer cells and promote cell apoptosis, and this dose combination can achieve the effect of high dose of TP.

In order to explore the mechanism of synergistic effect between the two doses, we conducted bioinformatics analysis compared the transcriptome of the monotherapy group and the combination therapy group using a blank control as a reference, we found that compared with monotherapy, two pathways closely related to cell survival, JAK-STAT signaling pathway and mTOR signaling pathway, appeared. Research has shown that regulating the JAK-STAT signaling pathway can inhibit the proliferation, migration, and invasion of liver cancer cells, and promote cell apoptosis[44-46]. Meanwhile, overactivation of mTOR signaling can promote liver cell carcinogenesis, especially in liver cell recovery and liver transplantation, and further promote liver cancer cell metastasis[47-49]. In this study, transcriptome analysis revealed that two monomers did not activate either of these two pathways in their respective individual measurements. However, when applied simultaneously, both pathways were activated simultaneously. By contrast, many natural small molecules such as quercetin and TP display polypharmacology, applying concurrent moderate pressure on multiple nodes that may dampen escape signaling and reduce rebound at clinically tolerable exposures.

In addition, this drug combination can reduce the dosage of individual drugs, which can alleviate various drug side effects. This idea is similar to the cocktail therapy, and there are other different combinations used to treat different tumors, such as osteosarcoma, non-small cell lung cancer, and breast cancer[50-53]. This seeks monomers from TCM, and through their different categories and dosage combinations, can provide more potential options for tumor treatment. Finally, we acknowledge that the precise translation of our optimal in vitro concentration ratio to the in vivo setting remains a limitation due to the complex pharmacokinetics of natural compounds, particularly the low oral bioavailability of quercetin and the challenge of achieving a uniform drug concentration within the solid tumor microenvironment, underscoring the need for advanced delivery strategies to facilitate clinical translation.

CONCLUSION

Although significant progress has been made in the treatment of liver cancer, it is still a refractory cancer with a high mortality rate. There are many active ingredients in TCM that have shown good anti liver cancer effects, among which TP and quercetin have good anti-liver cancer effects. However, single monomers often have limited anti-cancer effects or high toxic side effects, which limit their use. This study takes TP and quercetin as examples to seek the optimal synergistic effect dosage and achieve the maximum therapeutic effect. This ratio approach can be applied to two or even multiple chemical monomers, and the optimal ratio measurement activity can further enhance their application effectiveness and potential, providing a different way of thinking for the treatment of liver cancer and other types of tumors.

ACKNOWLEDGEMENTS

We thank all those who provided excellent technical support and assistance during the study.