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Introduction

China has a high incidence of esophageal cancer, with >95% of cases being squamous cell carcinoma (1,2). Esophageal squamous cell carcinoma (ESCC) develops slowly and presents with no obvious clinical symptoms in the early stages. Most patients present with advanced or locally advanced stages of ESCC when they are diagnosed, eliminating the possibility of surgery (3). The early detection rate of esophageal cancer has improved in recent years as China's medical level has advanced; however, the postoperative recurrence and metastasis rates remain high and the 5-year survival rate is only 30%, posing a serious threat to health (4,5). Platinum complexes are a class of chemotherapeutic drugs commonly used in clinical practice, which form DNA adducts by cross-linking with DNA strands, thus causing DNA damage and cytotoxicity (6). Cis-diamminedichloroplatinum (CDDP) was the first platinum-based chemotherapeutic drug formally used in the clinic and it is still frequently used due to its high efficacy and inexpensive cost (7). CDDP-based chemotherapy has evolved into a significant component of the comprehensive treatment strategy for ESCC, serving as the primary choice for postoperative adjuvant therapy and the first-line treatment for unresectable or recurrent ESCC (8). However, most patients encounter the clinical obstacle of drug resistance, which severely limits clinical efficacy (9); therefore, identifying effective medications that improve tumor CDDP resistance may markedly improve patient survival.

Herbal monomers possess antitumor properties, which can lessen the adverse effects of chemotherapy, increase treatment efficacy and improve the quality of life of patients (10). Triptolide (TPL), which is an extensively researched monomer of the Chinese anticancer medicinal tretinoin extract, was initially revealed to be a valuable medication in the treatment of rheumatoid arthritis. TPL has recently been discovered to have a strong inhibitory effect on a variety of tumor cells and solid tumors, including colon, liver, gastric, pancreatic, breast and ovarian cancer, which is linked to a number of mechanisms, including cell proliferation inhibition, apoptosis induction, antitumor invasion and anti-metastasis (11,12). TPL may also have a role in CDDP-induced DNA damage and repair and may improve tumor resistance to radiation. Fanelli et al (13) show that the combined use of CDDP and TPL could effectively limit DNA repair, overcoming the resistance of osteosarcoma to CDDP. Zhu et al (14) discovered that TPL combined with CDDP inhibits the growth of gemcitabine-resistant pancreatic cancer PANC-1 and MIA PaCa-2 cells in vivo and in vitro. Hu et al (15) and Huang et al (16) reveal that TPL markedly reduces the proliferation of CDDP-resistant human epithelial ovarian cancer cells, enhances cell sensitivity to CDDP and accelerates apoptosis. TPL has also recently been found to act as an effective regulator of glycolysis and mitochondrial activity, following extensive research into its mechanism of action (17–19). Notably, TPL has been shown to block mitochondrial hexokinase II (HK2) expression and aerobic glycolysis, resulting in mitochondrial translocation of the BAX/BAD complex and cleavage of the tumor suppressor GSDME by active cysteine 3, which can trigger head and neck cancer cellular pyroptosis (20). Furthermore, TPL can stimulate nasopharyngeal carcinoma cell apoptosis by inducing the degradation of the EBV-associated tumor antigen EBNA1 through increased susceptibility to caspase-9-dependent mitochondrial apoptosis (21). TPL has also been shown to markedly reduce ESCC progression (22,23). However, the effects of TPL on CDDP resistance, glycolysis and mitochondrial function in ESCC cells remain unknown.

The two primary routes for energy synthesis in cells are glycolysis and oxidative phosphorylation (24); most cells can switch between these two pathways and adapt to changes in their environment. When oxygen supply is insufficient, glucose is converted to pyruvate, which is converted to lactate in the cytoplasm, with net production of protons and excretion into the extracellular medium resulting in acidification of the medium around the cell into CO2 and water in the mitochondria via the tricarboxylic acid cycle and the respiratory chain when oxygen is adequately supplied (25). The energy metabolism of cancer cells differs from that of normal cells, resulting in the Warburg effect, in which tumor cells receive energy in a primarily glycolytic manner in the presence of sufficient oxygen (26–28). Although aerobic glycolysis and its offshoots are less effective modes of energy acquisition than mitochondrial oxidative phosphorylation, they can boost biosynthesis, offer biological raw materials for rapid tumor cell proliferation and aid in tumor growth (26,29,30). Several studies have revealed that high aerobic glycolysis rates and mitochondrial metabolism are linked to tumor development, progression and treatment resistance (31–34). Aurora-A can enhance CDDP resistance by decreasing cellular senescence and inducing glycolytic metabolism in ovarian cancer-like organs and cells (35), whereas promoting mitochondrial reactive oxygen species (ROS) generation and triggering cell death in rectal cancer cells can improve chemotherapeutic effectiveness (36). Elucidating the condition of glycolysis and mitochondrial energy metabolism is critical for understanding how CDDP resistance can be overcome in CDDP-resistant ESCC cells.

Based on the previously reported studies, the present study used CDDP-resistant TE1/CDDP and KYSE30/CDDP ESCC cells as research subjects to determine whether TPL can inhibit the growth of drug-resistant ESCC by inhibiting cellular glycolysis and inducing cellular mitochondrial damage. To assess this, the Cell Counting Kit (CCK)-8 assay, flow cytometry, wound healing assay, Transwell assay and nude mouse subcutaneous graft tumor experiments were performed. The present study may contribute to a better understanding of the anti-drug-resistant action of TPL in ESCC and could provide new therapeutic options for CDDP-resistant ESCC.

Materials and methods

Construction of CDDP-resistant ESCC cells

CDDP-resistant cells were generated using the incremental drug concentration approach (37) with ESCC cell lines (KYSE30 and TE1) obtained from the Cell Center of Shanghai Academy of Biological Sciences, Chinese Academy of Sciences. CDDP (cat. no. HY-17394; MedChem Express) was diluted to 0.2 µM in RPMI-1640 medium (cat. no. R0883; MilliporeSigma) containing 10% fetal bovine serum (cat. no. F0193; MilliporeSigma) and ESCC cells were grown for 48 h at 37°C in a 5% CO2 incubator. Subsequently, the cells were rinsed three times with PBS and the culture medium (without CDDP) was changed to continue the culture. The number and condition of the cells were examined using a light microscope and the cells were passaged when the density of adherent cell growth exceeded 80%. After passaging, the cells were cultured in the same medium containing 1.2 times the previous CDDP concentration. This procedure was repeated until ESCC cells could be maintained in the medium with a CDDP concentration of 10 µM, resulting in CDDP-resistant TE1/CDDP and KYSE30/CDDP cells.

Cell viability assay

KYSE30 and TE1 cells were digested with trypsin. After centrifugation at 300 × g, 4°C for 5 min, centrifuged and counted and inoculated into 96-well plates at 1×104 cells/well; 200 µl culture solution (containing 0, 1, 2, 4, 8, 16 and 32 µM CDDP) was added to each well and the plates were cultured at 37°C in a 5% CO2 incubator for 24 h. Subsequently, 20 µl CCK-8 solution (cat. no. HY-K0301; MedChem Express) was added to each well, mixed and incubated for 2 h. The OD value of the cells in each well at 450 nm wavelength was measured using an microplate reader. The IC50 values were estimated, as well as the resistance index of TE1/CDDP and KYSE30/CDDP cells to CDDP (resistance index=IC50 of resistant cells/IC50 of parental cells).

Similarly, KYSE30 and TE1 cells were treated with 0, 1, 2, 4, 8, 16 and 32 nM TPL and the IC50 values in TE1/CDDP and KYSE30/CDDP cells were computed to determine the best TPL treatment concentration (2 nM). TE1/CDDP and KYSE30/CDDP cell treatment was continued with varying CDDP concentrations in combination with 2 nM TPL and the IC50 values were calculated as aforementioned.

5-ethynyl-2′-deoxyuridine (EdU) assay for cell proliferation

The proliferation of TE-1/CDDP and KYSE30/CDDP cells was determined using the EdU kit (cat. no. C10310-1; Guangzhou RiboBio Co., Ltd.). Cells were inoculated in 24-well plates at 2×104 cells/well and treated with 2 nM TPL, 4 µM CDDP or 2 nM TPL + 4 µM CDDP for 24 h. The culture medium was then discarded and the cells were rinsed in PBS and stained with 10 µM EdU solution for 1 h at room temperature and in the dark. The cells were rinsed again in PBS and were then fixed and permeabilized using 4% paraformaldehyde and 0.3% Triton X-100 for 15 min at room temperature, respectively. Subsequently, the cells were incubated with Click reaction solution for 30 min at room temperature in the dark, before the nuclei were stained with DAPI for 10 min at room temperature, the slides were sealed and images were observed and captured under a fluorescence microscope (magnification, ×200; five fields of with uniform fluorescence distribution randomly observed). The EdU-positive cells were analyzed using ImageJ v1.8.0 software (National Institutes of Health), and the level of cell proliferation was determined.

Clone formation assay

The cells were randomly divided into four groups and were inoculated in Petri dishes containing 10 ml culture medium at a density of 100 cells/plate. The cell groups were treated with 2 nM TPL, 4 µM CDDP or 2 nM TPL + 4 µM CDDP, before being placed in an incubator at 37°C with 5% CO2 to continue incubation for 48 h. The colonies were fixed with 4% paraformaldehyde for 20 min at room temperature, then rinsed twice with PBS before being stained 30 min at room temperature with 0.1% crystal violet staining solution (cat. no. C0121; Beyotime Institute of Biotechnology), washed with PBS and air-dried. Images of the cells were captured under a light microscope (magnification, ×200) and the number of clones containing >50 cells was counted.

Flow cytometry

Following various treatments, TE1/CDDP and KYSE30/CDDP cells were collected and inoculated in cell culture dishes at 1×105 cells/ml. PI and Annexin V-FITC staining was performed at room temperature using the PI/Annexin V-FITC kit (cat. no. 40302ES20; Shanghai Yeasen Biotechnology Co., Ltd.) according to the manufacturer's instructions (incubated in the dark for 15 min). After adding 400 µl binding buffer, the staining was detected using a 2010284AA Flow Cytometer (Agilent Technologies, Inc.) and the apoptosis rate (the percentage of early + late apoptotic cells) was estimated using FlowJo v10 software (FlowJo LLC).

Cell cycle progression (cat. no. C1052, Beyotime Institute of Biotechnology) and ROS levels (cat. no. 50101ES01; Shanghai Yeasen Biotechnology Co., Ltd.) were measured respectively. The PI staining solution and ROS staining solution (10 µM DCFH-DA) were prepared as per the kit instructions. The two staining solutions were mixed with each group of cells (1×106), incubated in a 6-well plate at 37°C for 30 min in the dark (mixing every 5 min by inverting), centrifuged at 300 × g for 5 min at 4°C to remove the supernatant and washed and resuspended in PBS. The cells were filtered to produce a single-cell solution for flow cytometric analysis. Fluorescence intensity was recorded at an excitation wavelength of 488 nm and FlowJo v10 software (FlowJo LLC) was used to examine cell changes in cell cycle progression and ROS content.

Hoechst 33258 staining

TE1/CDDP and KYSE30/CDDP cells were treated with 2 nM TPL, 4 µM CDDP and 2 nM TPL + 4 µM CDDP. Fluorescence was detected using the Hoechst 33258 kit (Shanghai Yeasen Biotechnology Co., Ltd.). According to the manufacturer's instructions, 5 µM Hoechst 33258 staining solution was prepared in PBS and 100 µl staining solution was added dropwise to 96-well plates containing TE1/CDDP and KYSE30/CDDP cells. The cells were then incubated at 37°C for 20 min, after which the plates were washed twice with PBS. The staining was visualized with a fluorescence microscope (Leica Microsystems GmbH).

Wound healing assay

TE1/CDDP and KYSE30/CDDP cells were digested with trypsin, resuspended in PBS and inoculated in 6-well plates with labeled lines at 5×105 cells/well. When the cell growth density reached 80%, a 200-µl sterile pipette tip was used to draw a straight line perpendicular to the bottom of the well. The detached cells were washed away with pre-cooled PBS solution and 2 ml FBS-free RPMI-1640 medium was added to the culture. The cells were examined and images were captured under a microscope at 0 and 24 h. The width of the scratches was measured using ImageJ v1.8.0 software and relative migration rate was calculated using the following formula: (0 h migration spacing −24 h migration spacing)/0 h migration spacing ×100%.

Transwell detection

An 8-µm Transwell system (Corning, Inc.) was placed in a 24-well plate and 50 µl Matrigel (cat. no. 354234; Corning, Inc.) was added and air-dried for 4 h at room temperature. Subsequently, 100 µl TE1/CDDP and KYSE30/CDDP cell suspensions at a density of 1×105/ml were added to the chambers and 500 µl RPMI-1640 medium containing 10% FBS was added to each well underneath the chambers. The plates were then incubated at 37°C in a 5% CO2 incubator. After removing the plate, the media from the upper and lower chambers were discarded and the cells in the upper chamber were gently removed with a cotton swab. The cells were then fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet (cat. no. C0121; Beyotime Institute of Biotechnology) for 30 min at room temperature, washed twice with PBS and air-dried and then observed and photographed under a high-magnification microscope magnification, ×200; five fields of with uniform fluorescence distribution randomly observed). The number of invasive cells detected under the microscope was counted using ImageJ v1.8.0 software.

Detection with kits

The reagents and samples (TE1/CDDP and KYSE30/CDDP cell supernatants) were prepared according to the instructions of the kits (all from Nanjing Jiancheng Bioengineering Institute) on indicators including the glucose uptake level (cat. no. A154-1-1), lactic acid production (cat. no. A019-2-1), glutathione (GSH) level (cat. no. A006-2-1) and superoxide dismutase (SOD) level (cat. no. A001-1-2). Indicators were all detected using the colorimetric method. The absorbance of the cells was then measured using an enzyme labeling instrument (cat. no. DR-200Bc; Diatek) and the concentration of each index was estimated using the standard curve.

Extracellular acidification rate (ECAR) detection

ECAR is a useful indicator for assessing the acid produced by anaerobic fermentation during cell metabolism and its change can represent cell metabolism and growth status, whereas a fall in pH will reduce the fluorescence intensity of the probe (38). In the present study, ECAR was detected using the Seahorse XF Glycolytic Stress Test Kit (cat. no. 103020-100; Agilent Technologies, Inc. Santa Clara, CA, USA) and Seahorse XFe24 Analyzer. Cells were treated with 2 nM TPL, 4 µM CDDP and 2 nM TPL + 4 µM CDDP for 24 h at 37°C, according to the experimental design. Subsequently, trypsin was used to digest TE1/CDDP and KYSE30/CDDP cells, which were washed and collected in PBS before being centrifuged (300 × g, 4°C for 5 min) and resuspended. The experiment involved inoculating 1×104 cells in 96-well culture plates, as per the protocol. Cells were grown in XF basic media without glucose and pyruvate at 37°C. Glucose (10 mM), oligomycin (1 µM) and 2-DG (50 mM) were introduced consecutively at particular time points according to the directions for use. ECAR data were obtained and displayed using the Seahorse XF24 software (Agilent Technologies, Inc.).

ATP content detection

The ATP Assay Kit (cat. no. S0026; Beyotime Institute of Biotechnology) was used to detect ATP content. According to the manufacturer's instructions, the standard curve and ATP working solution were prepared. After various treatments, TE1/CDDP and KYSE30/CDDP cells were lysed using lysis solution. The supernatant was collected by centrifugation at 300 × g for 5 min (4°C) and resuspended and 100 µl ATP working solution was applied to a 6-well plate. Subsequently, 20 µl cell supernatant was added to each well and a chemiluminescence analyzer (cat. no. 2805880; Thermo Fisher Scientific, Inc.) was used to quickly detect the chemiluminescence intensity of each solution. The mitochondrial ATP content of cells in each group was determined using a standard curve and the relative expression of the control group was analyzed.

Mitochondrial membrane potential detection

Following various treatments, TE1/CDDP and KYSE30/CDDP cells were washed with PBS. According to the directions of the Mitochondrial membrane potential assay kit (cat. no. C2006, Beyotime Institute of Biotechnology), culture medium and JC-1 staining working solution were added to 6-well plates in a 1:1 volume ratio, carefully mixed and incubated at 37°C for 20 min in the dark. The supernatant was then removed and the cells were washed twice with the prepared staining buffer to eliminate any JC-1 probe that had not attached to the cells. Subsequently, 2 ml cell culture medium was added, the results were observed under a fluorescence microscope and images were captured. Elevated membrane potential indicated polarization, with JC-1 aggregates exhibiting red fluorescence and reduced membrane potential indicates depolarization, with JC-1 monomers exhibiting green fluorescence.

Animal experiments

Shanghai Model Organisms Center, Inc. provided 20 5-week-old male BALB/c nude mice, which were randomly divided into the following four groups each containing five mice: Control, TPL, CDDP and TPL + CDDP. Mice were housed under a 12-h light/dark cycle at a temperature of 22–26°C and relative humidity of 50–60%. To create a tumor xenograft model, 3×106 KYSE30/CDDP cells (100 µl) were implanted into the right axilla of nude mice. After 7 days of feeding, mice in the TPL group were administered TPL intraperitoneally at 0.45 mg/kg/time. Mice in the CDDP group were administered 1.5 mg/kg/time CDDP intraperitoneally. In the TPL + CDDP group, mice were administered 0.45 mg/kg/time TPL + 1.5 mg/kg/time CDDP by gavage. All treatments were administered every 3 days for five consecutive times, whereas the control group received saline by gavage. Tumor volume was determined every 7 days in mice using the following formula: Volume (mm3)=1/2 × L × W2 (where L represents tumor length and W represents tumor width). The mice were maintained for 28 days before being cervically dislocated and sacrificed, with the tumors carefully isolated, weighed and photographed. The experimental nude mice in the present study all weighed ~25 g. According to the guidelines for the review of humane endpoints in animal experiments (RB/T 173–2018) (39), the mouse tumors should not be larger than 17 mm and their mass should not exceed 1.25 g (5% of body weight). The present study was approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University (Henan, China; approval no. 20220096).

TUNEL apoptosis assay

Apoptosis in tumor tissues was assessed using a TUNEL kit (cat. no. C1091; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Mouse tumors were fixed in 4% paraformaldehyde (4°C for 24 h), dried, embedded in paraffin and cut into 4-µm sections. Tumor tissue sections were deparaffinized with xylene, hydrated with a gradient series of ethanol and then treated with 20 µg/ml DNase-free proteinase K solution at room temperature for 30 min to promote reaction reagents to enter the nucleus. After washing with PBS, tissue sections were submerged in 3% H2O2 for 15 min to deactivate the endogenous peroxidases. The sections were then rinsed with PBS and stained with 50 µl TUNEL staining solution (cat. no. C1005; Beyotime Institute of Biotechnology) at 37°C for 60 min, before being washed with PBS and incubated with DAPI staining solution (cat. no. C1005; Beyotime Institute of Biotechnology) at room temperature in the dark for 5 min. The sections were then blocked with anti-fluorescence burst sealing solution and the percentage of TUNEL-positive cells was calculated under a microscope.

Immunohistochemistry

Tissue sections were dehydrated with ethanol, underwent antigen retrieval with citrate (cat. no. C1032; Beijing Solarbio Science & Technology Co., Ltd.) and were blocked with avidin/biotin blocking buffer (cat. no. C-0005; Shanghai Haoran Biotechnology Co., Ltd.) at room temperature before being incubated with Ki67 (1:200; cat. no. ab232784; Abcam), cleaved caspase-3 (1:100; cat. no. PA5-114687; Thermo Fisher Scientific, Inc.) and cleaved caspase-9 (1:100; cat. no. PA5-105271; Thermo Fisher Scientific, Inc.) primary antibodies at 4°C overnight. After washing, the appropriate secondary antibody (1:500; cat. no. ab150077; Abcam) was applied to the tissue sections and incubated for 1 h at room temperature. The sections were then stained for five min at room temperature with streptavidin-horseradish peroxidase and hematoxylin (cat. no. C0107, Beyotime Institute of Biotechnology) and underwent dehydration in an ethanol concentration gradient, permeabilization with xylene and section sealing with neutral gum. The sections were observed under a light microscope (magnification, ×200) and images were captured.

Western blot analysis

TE1/CDDP and KYSE30/CDDP cells, as well as tumor tissues, were collected following various treatments and RIPA lysis buffer was added to extract total proteins. The protein content in the supernatant was measured using the BCA Protein Kit (cat. no. P0012; Beyotime Institute of Biotechnology). A 10% SDS-PAGE gel was then prepared and protein samples were loaded at 20 µg/well for electrophoresis. The proteins were subsequently transferred to a PVDF membrane and blocked with 5% bovine serum albumin (cat. no. ST025; Beyotime Institute of Biotechnology) for 1 h at room temperature. Afterwards, rabbit anti-cyclin-dependent kinase 4 (CDK4; 1:2,000; cat. no. ab199728), cyclin D1 (1:100; cat. no. ab16663), Bcl-2 (1:2,000; cat. no. ab182858), cleaved caspase-3 (1:500; cat. no. ab32042), cleaved caspase-9 (1:1,000; cat. no. 9505; CST, MA, USA), E-cadherin (1:1,000; cat. no. ab212059), Vimentin (1:1,000; cat. no. ab137321), Snail (1:1,000; cat. no. ab216347), glucose transporter protein 1 (GLUT1; 1:100,000; cat. no. ab115730), HK2 (1:1,000; cat. no. ab209847), lactate dehydrogenase A (LDHA; 1:1,000; cat. no. 2012S; CST), cytochrome c (Cytc; 1:5,000; cat. no. ab133504), COX IV (1:2,000; cat. no. ab202554) and β-actin (1:1,000; cat. no. ab8227) primary antibodies were added according to the experimental design and incubated at 4°C overnight. After washing with TBST (0.05% Tween), sheep anti-rabbit IgG (1:2,000; cat. no. ab6721) was added and incubated at room temperature for 30 min. With the exception of the cleaved caspase-9 and LDHA antibodies, all antibodies were purchased from Abcam. Cytc was measured using the Cellular Mitochondrial Isolation Kit (cat. no. C3601; Beyotime Institute of Biotechnology), which extracted mitochondrial proteins while eliminating cytoplasmic proteins. After washing with TBST, ECL chemiluminescent solution (cat. no. P0018S; Beyotime Institute of Biotechnology) was added dropwise to develop the color and exposure. Images of the protein bands were captured with a chemiluminescent image analysis system (5200; Tanon, Shanghai, China) and the grayscale values of the protein bands of each group were semi-quantitatively analyzed using ImageJ v1.8.0 software (National Institutes of Health).

Statistical analysis

The experimental data were analyzed using SPSS 26.0 software (IBM Corp.) and are presented as mean ± standard deviation. To analyze differences between the groups, a Student's t-test (unpaired) or one-way ANOVA with Tukey's post hoc test was performed. P<0.05 was considered to indicate a statistically significant difference.

Results

TPL increases the inhibitory effect of CDDP on the proliferation of CDDP-resistant ESCC cells

CDDP was used to treat drug-resistant cells and their parental cells at various concentrations (0, 1, 2, 4, 8, 16 and 32 µM). Cell viability decreased with increasing CDDP treatment time, as measured by CCK-8 assay. The IC50 values of CDDP in TE-1 and KYSE30 cells were 3.94 and 3.07 µM, respectively and those in TE1/CDDP and KYSE30/CDDP cells were 19.71 and 14.57 µM, respectively; the resistance index was 5.00 and 4.75 (Fig. 1A and B), indicating successful construction of CDDP-resistant cell lines. Based on the literature, a CDDP action concentration of 4 µM was established. As shown in Fig. 1C, TE1/CDDP and KYSE30/CDDP cells were further treated with 0, 1, 2, 4, 8, 16 and 32 nM TPL and it was discovered that cell viability gradually decreased with increasing TPL treatment concentration, with no significant difference in the effect of TPL on the two drug-resistant cell lines. The experimental dose of TPL (cell viability ≥70%) was determined to be 2 nM, which was the highest inhibitory concentration without cytotoxicity. After treating resistant cells with different concentrations of CDDP in combination with 2 nM TPL, the IC50 values in TE1/CDDP and KYSE30/CDDP cells were significantly reduced compared with CDDP alone (Fig. 1D), demonstrating that the addition of TPL increased the inhibitory effects of CDDP on the viability of CDDP-resistant ESCC cells. The subsequent tests established CDDP and TPL treatment doses at 4 µM and 2 nM, respectively. EdU and clone formation experiments both revealed that the combination of the two inhibited the proliferation of TE1/CDDP and KYSE30/CDDP cells more effectively than CDDP and TPL alone and the proportion of EdU-positive cells and the number of cloned cells were found to be even lower (Fig. 2E-I). Taken together, the experimental results revealed that TPL considerably increased the inhibitory effects of CDDP on the proliferation of CDDP-resistant ESCC cells.

Figure 1. TPL increases the proliferative effect of CDDP on anti-CDDP-resistant ESCC cells. (A and B) The cells viability was measured with CCK-8. (C) The effects of various TPL concentrations on the proliferative viability of TE-1/CDDP and KYSE30/CDDP cells were detected using CCK-8. (D) 2 nM TPL was administered concurrently with CDDP treatment of TE-1/CDDP, KYSE30/CDDP cells and viability was determined by CCK-8. (E and F) EdU detection of proliferation in TE-1/CDDP and KYSE30/CDDP cells (magnification, ×20; scale bar 100 µm). (G-I) Clone formation assay to determine the formation levels of TE-1/CDDP and KYSE30/CDDP cells. *P<0.05. TPL, triptolide; CDDP, cis-diamminedichloro-platinum; ESCC, esophageal squamous cell carcinoma; CCK, cell counting kit; TE-1/CDDP, cisplatin-resistant TE-1 cells; KYSE30/CDDP, cisplatin-resistant KYSE30 cells; IC50, median inhibition concentration; DAPI, diamidino-phenyl-indole; EdU, 5-ethynyl-2′-deoxyuridine.

Figure 2. TPL enhances CDDP-promoted CDDP-resistant ESCC cell cycle blockade. (A-C) Cell cycle arrest in TE-1/CDDP and KYSE30/CDDP cells was observed using a flow cytometric assay under various treatment conditions. (D-F) Differences in the level of cell cycle proteins in TE-1/CDDP and KYSE30/CDDP cells under various treatment conditions. *P<0.05. TPL, triptolide; CDDP, cis-diamminedichloro-platinum; ESCC, esophageal squamous cell carcinoma; TE-1/CDDP, cisplatin-resistant TE-1 cells; KYSE30/CDDP, cisplatin-resistant KYSE30 cells; CDK4, cyclin-dependent kinase 4.

TPL promotes the effects of CDDP on CDDP-resistant ESCC cell cycle arrest

The present study investigated the effect of TPL on CDDP-mediated cell cycle arrest in CDDP-resistant ESCC cells using flow cytometry. The results showed that when CDDP and TPL were used to treat cells alone, the number of TE1/CDDP and KYSE30/CDDP cells in the G0/G1 phase was significantly elevated, whereas the number of cells in the S phase was significantly decreased compared with the control group. The combination of CDDP and TPL further accelerated the accumulation of cells in the G0/G1 phase (Fig. 2A-C), implying that TPL may enhance the effects of CDDP on the cell cycle arrest of CDDP-resistant ESCC cells. Cyclin D1 and CDK4 play critical roles in cell cycle control and proliferation (40) and the expression of cyclin D1 and CDK4 can be evaluated to determine cell cycle progression. The results of western blot analysis revealed that CDDP and TPL significantly inhibited the expression of cyclin D1 and CDK4 in TE1/CDDP and KYSE30/CDDP cells and CDDP and TPL had a synergistic effect, preventing CDDP-resistant ESCC cells from entering the S phase for cell proliferation (Fig. 2D-F). TPL was observed to enhance CDDP-mediated G0/G1 phase arrest.

TPL promotes the CDDP-induced apoptosis of CDDP-resistant ESCC cells

The effects of TPL on CDDP-mediated cytotoxicity in TE1/CDDP and KYSE30/CDDP cells were further investigated. As demonstrated by inverted fluorescence microscopy, Hoechst 33258 staining revealed that the morphology of CDDP-resistant ESCC cells was markedly changed after CDDP and TPL treatment. As shown in Fig. 3A-C, the nuclei of the two drug-resistant cells in the control group exhibited light blue fluorescence and the blue fluorescence of the nuclei was enhanced after treatment with CDDP and TPL alone; however, the nuclei of the cells in the group co-treated with CDDP and TPL showed a bright blue color and the morphology of the cells showed chromatin margin clustering, karyopyknosis, nuclear fragmentation and apoptotic body formation. These findings suggested that TPL may enhance the effects of CDDP and induce the apoptosis of CDDP-resistant ESCC cells. The rates of apoptosis measured by flow cytometry showed a consistent trend (Fig. 3D-F).

Figure 3. TPL enhances CDDP pro-apoptotic effects in CDDP-resistant ESCC cells. (A-C) The apoptotic levels of TE-1/CDDP and KYSE30/CDDP cells were measured under each treatment condition using Hoechst 33258 fluorescent staining (magnification, ×40; scale bar, 50 µm). (D-F) Flow cytometry test for detecting apoptotic changes in TE-1/CDDP and KYSE30/CDDP cells under various treatment conditions. (G-I) Western blotting was used to determine the expression levels of apoptotic proteins in TE-1/CDDP and KYSE30/CDDP cells under various treatment conditions. *P<0.05. TPL, triptolide; CDDP, cis-diamminedichloro-platinum; ESCC, esophageal squamous cell carcinoma; TE-1/CDDP, cisplatin-resistant TE-1 cells; KYSE30/CDDP, cisplatin-resistant KYSE30 cells.

The apoptosis markers Bcl-2, cleaved caspase-3 and cleaved caspase-9 were detected using western blotting. The results showed that the combination of CDDP and TPL further inhibited Bcl-2 and promoted the levels of cleaved caspase-3 and cleaved caspase-9 proteins compared with the treatments alone (Fig. 3G-I). These findings indicated that TPL could enhance the pro-apoptotic effects of CDDP on TE1/CDDP and KYSE30/CDDP cells.

TPL increases the inhibitory effect of CDDP on CDDP-resistant ESCC cell migration, invasion and epithelial-mesenchymal transition (EMT)

The present study investigated CDDP-resistant ESCC cells using cell scratch and Transwell assays. The outcomes revealed that the CDDP combined with TPL-treated group had a significantly lower relative migration rate and quantity of cells entering the lower compartment than the treatment groups alone (Fig. 4A-F). The protein levels of EMT-related markers, E-cadherin, Vimentin and Snail, were measured utilizing western blotting (Fig. 4G-I). The levels of the epithelial marker E-cadherin were increased in CDDP-resistant ESCC cells in the TPL + CDDP group compared with in the treatment groups alone, whereas the expression levels of the mesenchymal marker Vimentin and EMT-inducing transcription factor Snail were decreased. These findings demonstrated that TPL could improve the sensitivity of CDDP-resistant ESCC cells to CDDP and inhibit the occurrence of EMT, thus suppressing the invasive and metastatic ability of the cells.

Figure 4. TPL enhances CDDP inhibition of CDDP-resistant ESCC cell migration, invasion and EMT effects. (A-C) The migration levels of TE-1/CDDP and KYSE30/CDDP cells after treatment with TPL and CDDP were determined using a scratch test (magnification, ×10; scale bar 200 µm). (D-F) The Transwell assay was used to determine the quantity of TE-1/CDDP and KYSE30/CDDP cells that invaded the lower chamber following TPL and CDDP treatment (magnification, ×40; scale bar, 50 µm). (G-I) Western blot analysis was used to determine the expression levels of EMT-related proteins in TE-1/CDDP and KYSE30/CDDP cells following TPL and CDDP therapy. *P<0.05. TPL, triptolide; CDDP, cis-diamminedichloro-platinum; ESCC, esophageal squamous cell carcinoma; EMT, epithelial-mesenchymal transition; TE-1/CDDP, cisplatin-resistant TE-1 cells; KYSE30/CDDP, cisplatin-resistant KYSE30 cells.

TPL increases the sensitivity of CDDP-resistant ESCC cells to CDDP by suppressing glycolysis

One of the characteristics of tumor cells is the reprogramming of energy metabolism through glycolysis and mitochondrial oxidative phosphorylation. The present study initially assessed the effect of each treatment condition on glycolysis in CDDP-resistant ESCC cells. As shown in Fig. 5A-D, TPL combined with CDDP treatment inhibited cellular glucose uptake and lactate production more than CDDP alone, implying that TPL may further decrease glycolysis in CDDP-resistant ESCC cells. When cellular glycolysis creates lactate and excretes it into the environment, protons are also ejected, resulting in extracellular acidification, making ECAR a useful diagnostic marker for detecting glycolysis. To investigate cellular glycolysis, ECAR was evaluated. The results of normalization calculations are displayed in Fig. 5E and F and all four cell groups showed the same trend of change. The ECAR values of TE1/CDDP and KYSE30/CDDP cells rose after the injection of a saturating concentration of glucose, reflecting the cellular glycolytic capability at basal conditions. Further injection of oligomycin inhibited oxidative phosphorylation, allowing cellular glycolysis to achieve its full capacity and ECAR to rise to its peak. The subsequent injection of 2-DG hindered glycolysis and ECAR readings markedly dropped. The CDDP-resistant ESCC cells in the TPL and CDDP-treated groups had considerably lower ECAR values than the control group. The combination therapy reduced proton efflux and ECAR values, suggesting that TPL hindered cellular glycolysis. The expression levels of key glycolytic genes GLUT1, HK2 and LDHA were detected by western blotting under different treatment conditions, as shown in Fig. 5G-I. TPL combined with CDDP suppressed the levels of these factors more efficiently, indicating the inhibitory impact of TPL on cellular glycolysis, which may be an important cause of enhanced CDDP sensitivity in CDDP-resistant ESCC cells.

Figure 5. TPL enhances CDDP sensitivity of CDDP-resistant ESCC cells via inhibiting glycolysis. (A-D) Kits were used to detect glucose absorption and lactate generation in TE-1/CDDP and KYSE30/CDDP cells from each group. (E and F) Kits were used to detect ECAR in TE-1/CDDP and KYSE30/CDDP cells from each group in order to assess cellular energy metabolic activity. (G-I) Western blot analysis revealed changes in the levels of key glycolysis regulators GLUT1, HK2 and LDHA in TE-1/CDDP and KYSE30/CDDP cells from each group. *P<0.05. TPL, triptolide; CDDP, cis-diamminedichloro-platinum; ESCC, esophageal squamous cell carcinoma; TE-1/CDDP, cisplatin-resistant TE-1 cells; KYSE30/CDDP, cisplatin-resistant KYSE30 cells; GLUT1, glucose transporter type 1; ECAR, extracellular acidification rate; HK2, hexokinase 2; LDHA, lactate dehydrogenase A.

TPL increases the CDDP sensitivity of CDDP-resistant ESCC cells by generating mitochondrial dysfunction

The effect of TPL on mitochondrial oxidative phosphorylation was further investigated. The combined treatment of 2 nM TPL and 4 µM CDDP significantly lowered ATP levels in TE1/CDDP and KYSE30/CDDP cells, indicating poor mitochondrial energy metabolism (Fig. 6A). Mitochondria are the primary intracellular source of ATP and defective mitochondrial energy metabolism generates a considerable amount of ROS. The ROS fluorescent probe DCFH-DA was used to measure ROS levels in CDDP-resistant ESCC cells. As shown in Fig. 6B and C, DCFH-DA fluorescence signals in TE1/CDDP and KYSE30/CDDP cells were increased in response to TPL and CDDP treatment alone. Furthermore, the DCFH-DA fluorescence signal was significantly boosted after combining the two cell treatments. In addition, a kit was used to investigate changes in GSH and SOD concentration in the cells. GSH and SOD can inactivate ROS, thereby protecting mitochondria from free radicals, maintaining mitochondrial structure and function (41). The results showed that TPL combined with CDDP significantly decreased the levels of GSH and SOD compared with the two treatments alone (Fig. 6D and E), indicating that TPL exacerbated CDDP-induced mitochondrial dysfunction. The effect of TPL on the mitochondrial membrane potential of CDDP-resistant ESCC cells was detected using JC-1 probe dye and flow cytometry (Fig. 6F and G). TE1/CDDP and KYSE30/CDDP cells treated with 2 nM TPL + 4 µM CDDP showed a significant decrease in red fluorescence and an increase in green fluorescence compared with the treatment groups alone. This suggested a decrease in mitochondrial membrane potential, which may be one of the important causes of cell death. Cytc is an electron carrier in the mitochondrial electron transport chain that is released into the cytosol when the cell is injured, initiating the creation of apoptotic bodies and resulting in apoptosis (42,43). As shown in Fig. 6H-J, CDDP-resistant ESCC cells were treated with various conditions that resulted in a large amount of Cytc being released into the cytosol and the Cytc content in the cytosol and mitochondria increased and decreased, respectively, with the TPL + CDDP treatment group having the most significant effect. These findings indicated that TPL may inhibit glycolysis and produce mitochondrial dysfunction, which alters energy metabolism; this may be the primary explanation for its ability to increase CDDP sensitivity in CDDP-resistant ESCC cells.

Figure 6. TPL increases the CDDP sensitivity of CDDP-resistant ESCC cells via generating mitochondrial dysfunction. (A) The kit identified ATP generation in TE-1/CDDP and KYSE30/CDDP cells from each group. (B and C) The ROS generation levels of TE-1/CDDP and KYSE30/CDDP cells in each group were detected via by flow cytometry utilizing the DCFH-DA technique. (D and E) The kit measured the GSH and SOD levels in TE-1/CDDP and KYSE30/CDDP cells from each group. (F and G) Each group's TE-1/CDDP and KYSE30/CDDP cells were tested for mitochondrial membrane potential using the JC-1 assay (magnification, ×20; scale bar 100 µm). (H-J) Western blot analysis was used to assess cytochrome c levels in the mitochondria and cytoplasm of TE-1/CDDP and KYSE30/CDDP cells under various treatment conditions. *P<0.05. TPL, triptolide; CDDP, cis-diamminedichloro-platinum; ESCC, esophageal squamous cell carcinoma; TE-1/CDDP, cisplatin-resistant TE-1 cells; KYSE30/CDDP, cisplatin-resistant KYSE30 cells; GSH, glutathione; SOD, superoxide dismutase; Cytc, cytochrome c; COX IV, cytochrome c oxidase IV.

TPL increases the susceptibility of CDDP-resistant ESCC cells to CDDP in vivo

To determine whether the addition of TPL had similar effects on CDDP in vivo as it did in vitro, 5-week-old nude mice were divided into four groups: Control, TPL, CDDP and TPL + CDDP for the subcutaneous tumor transplantation experiment (Fig. 7A). KYSE30/CDDP cells were injected subcutaneously into the right abdomen of naked mice for 1 week before a subcutaneous tumor developed. After 21 days of various administration regimens, it was revealed that all administration groups effectively inhibited tumor volume and weight compared with in the control group (Fig. 7B-D). Notably, the combined treatment of TPL and CDDP had the highest inhibitory effect on tumor volume and weight compared with TPL and CDDP alone (Fig. 7B-D). TUNEL staining revealed that the proportion of positive apoptotic cells in tumor tissues was considerably higher in the TPL + CDDP group than in the groups administered TPL and CDDP alone, implying that TPL increased the apoptosis induced by CDDP administration (Fig. 7E). Furthermore, immunohistochemical staining revealed that the combination of TPL and CDDP markedly decreased the levels of the proliferative protein Ki-67 and promoted the levels of the apoptotic proteins cleaved caspase-3 and cleaved caspase-9 in the tumor tissues, compared with in the group administered TPL or CDDP alone (Fig. 7G). The levels of important glycolysis regulators GLUT1, HK2 and LDHA, as well as the ratio of mitochondrial red/green fluorescence, followed a similar pattern; i.e., control group>TPL group>CDDP group>TPL + CDDP group, with statistical significance detected across all groups (Fig. 7H-K). These findings suggested that TPL may inhibit the glycolysis of CDDP-resistant ESCC cells in vivo and generate a drop in mitochondrial membrane potential to promote apoptosis, which may be the primary reasons that TPL enhances the sensitivity of resistant cells to CDDP.

Figure 7. TPL increases the susceptibility of CDDP-resistant ESCC cells to CDDP in vivo. (A) Animal models were constructed according to the timeline. (B-D) Subcutaneous tumor volumes were measured using vernier calipers and the mice were killed on the 28th day of feeding, with the isolated tumors weighed and photographed. (E and F) Tissue sections from the isolated tumors were produced and apoptosis was identified using the Tunel method (magnification, ×40; scale bar, 50 µm). (G) Immunohistochemistry was used to identify the expression levels of Ki67, cleaved caspase-3 and cleaved caspase-9 in each group's tumor sections (magnification, ×40; scale bar, 50 µm). (H and I) Western blot analysis of tumor tissues from each group revealed the expression levels of major glycolysis regulators GLUT1, HK2 and LDHA. (J and K) The intensity of red/green fluorescence in tumor tissues from each group was measured using the JC-1 method to quantify mitochondrial membrane potential alterations (magnification, ×20; scale bar 100 µm). *P<0.05. TPL, triptolide; CDDP, cis-diamminedichloro-platinum; ESCC, esophageal squamous cell carcinoma; KYSE30/CDDP, cisplatin-resistant KYSE30 cells; DAPI, diamidino-phenyl-indole; GLUT1, glucose transporter type 1; HK2, hexokinase 2; LDHA, lactate dehydrogenase A.

Discussion

The efficacy and low toxicity of Chinese herbs make them highly favorable and potentially useful in clinical cancer treatment and they have become a research hotspot for several types of tumor immunotherapy (44). TPL, an epoxy diterpene lactone compound with multiple biological activities and a molecular weight of 360.4 g/mol, was first isolated and named in 1972 from the root of Tripterygium wilfordii and has been suggested as a promising candidate for the clinical treatment of tumors and autoimmune diseases (45). TPL overdose affects various tissues and organs in the body and causes substantial difficulties associated with toxicity, severely limiting its therapeutic use (12). As a result, the present study used the CCK-8 assay to investigate the effect of various concentrations of TPL on the viability of CDDP-resistant ESCC cells and the highest active concentration of TPL that did not cause cytotoxicity was identified (2 nM). At this concentration, the regulatory effects of TPL on the malignant growth of CDDP-resistant ESCC cells were evaluated, as well as its effects on glycolysis and mitochondrial metabolism, in order to demonstrate its function in CDDP sensitivity and to understand the molecular mechanisms.

One of the most distinguishing aspects of ESCC is its high invasiveness and metastatic potential. Cancer cells of epithelial origin can spread locally and regionally by EMT before entering arteries and resulting in distant metastasis; therefore, the presence of EMT in cancer cells is seen as a significant phase in tumor invasion and metastatic progression (46). In the present study, treatment with TPL and CDDP (particularly a combination of these therapies) markedly increased the epithelial marker E-cadherin, while decreasing the mesenchymal marker Vimentin and the EMT transcription factor Snail, indicating that the process of EMT was suppressed. The combined treatment of TPL and CDDP also significantly reduced the cell scratch migration rate and the number of cells invading the lower chamber of the Transwell system, indicating that TPL could impede the invasion and metastasis of CDDP-resistant ESCC cells by suppressing EMT. EMT has also been proven to alter tumor chemosensitivity. It has been observed that EMT causes resistance to chemotherapeutic drugs, such as tamoxifen, CDDP and paclitaxel, in breast cancer cells and that it is correlated with radioresistance in breast cancer tumors (47). Yu et al (48) reveal that baicalein could inhibit EMT and promote apoptosis in CDDP-resistant lung adenocarcinoma cells via the PI3K/Akt/NF-κB pathway; this increases the susceptibility of drug-resistant lung adenocarcinoma cells to CDDP. Zhao et al (49) found that microRNA-128-3p inhibits EMT by downregulating the levels of EMT-transforming proteins (c-Met, PDGFRα, Notch1 and Slug), making glioblastoma more sensitive to temozolomide treatment. The aforementioned research findings indicate that EMT plays a crucial role in tumor chemosensitivity that cannot be overlooked. In the present study, treatment with 2 nM TPL + 4 µM CDDP inhibited EMT in TE1/CDDP and KYSE30/CDDP cells, resulting in increased E-cadherin and decreased Vimentin and Snail expression. Under this condition, cell proliferation was suppressed as measured by EdU and clone formation assays and flow cytometry and western blot analysis detected elevated rates of apoptosis and severe G0/G1 cell cycle arrest, indicating that CDDP-resistant ESCC cells are more sensitive to CDDP. EMT was shown to be an essential component of acquired CDDP resistance in CDDP-resistant ESCC cells and TPL could efficiently reverse CDDP chemoresistance by suppressing the EMT process.

Tumor growth rate is a crucial determinant influencing patient prognosis and improper energy metabolism has an impact on tumor growth rate. Reversing the energy metabolism of tumor cells can make them more chemosensitive (50). One of the most common characteristics in tumor cells is elevated activation of aerobic glycolysis, which is known as a cancer hallmark (51). Cancer cell metabolism is largely dependent on glycolysis and even under normal oxygen concentrations and with fully functional mitochondria, most cancer cells produce energy via glycolysis, with the glycolytic pathway accounting for ~50% of ATP production; this is also known as the Warburg effect or aerobic glycolysis (52). This metabolic alteration causes larger amounts of lactic acid in the tumor microenvironment, resulting in tumor acidosis, which aids cancer cell adaption to hypoxic circumstances and promotes tumor development and invasiveness (53). The use of aerobic glycolysis or oxidative phosphorylation by cancer cells is determined by a number of parameters, including cancer cell stage, tumor microenvironment and cell growth rate. Rapidly reproducing tumor cells may use aerobic glycolysis to create ATP for intracellular anabolism, but slowly growing drug-resistant cancer cells are more likely to rely on oxidative phosphorylation for survival (54). In the present study, the viability of TE1/CDDP and KYSE30/CDDP resistant cells treated with different concentrations of CDDP was much higher than that of parental cells, implying that the energy metabolism of the two types of cells are not same, TE1/CDDP and KYSE30/CDDP cells may rely primarily on aerobic glycolysis to maintain their proliferation. After co-treatment with TPL and CDDP, CDDP-resistant ESCC cells proliferated at a slower rate, with considerably more cells in the G0/G1 phase than the S phase. In addition, the rate of extracellular acidification caused by glycolysis was reduced, mitochondrial ATP was depleted, oxidative stress ensued and substantial amounts of Cytc were released into the cytoplasm. These findings suggested that TPL combined with CDDP impaired both energy metabolism modes, which could be the primary explanation for the effects of TPL on the increased sensitivity of CDDP-resistant ESCC cells to CDDP. Wang et al (55) discovered that TPL blocks the nucleotide excision repair pathway in melanoma, increases tumor cell sensitivity to carboplatin in vitro and in vivo and decreases tumor development. Zhu et al (56) demonstrate that TPL increases the susceptibility of non-small cell lung cancer cells to CDDP, etoposide and epirubicin by blocking the Nrf2-ARE pathway, decreasing cancer cell proliferation and transplanted tumor growth in mice in vivo.

The present study also assessed the effects of TPL on tumor growth in CDDP-resistant mice in vivo. The results revealed that the tumor volume and mass in the TPL + CDDP group were significantly lower than those in the treatment only groups. After the tumor tissues were cut into sections, TUNEL and immunohistochemical staining were performed to assess apoptosis and proliferation and the results showed that compared with in the treatment groups alone, the apoptosis level (TUNEL-positive cells and cleaved caspase-3 and cleaved caspase-9 staining) of the sections in the TPL + CDDP combined treatment group was significantly upregulated, while the proliferation level (Ki67) was significantly downregulated, indicating that TPL had a sensitizing effect on CDDP-resistant ESCC tumors, inducing apoptosis and inhibiting cell proliferation. In addition, TPL hindered glycolysis and caused mitochondrial damage in vivo, as evidenced by alterations in major glycolysis regulators and mitochondrial membrane potential measurements. The present study elucidated the effect of TPL on CDDP-resistant cells using in vivo and in vitro experiments. In light of the current findings, it may be hypothesized that TPL has the potential to be a therapeutic medication for the treatment of chemotherapy-resistant malignant tumors.

TPL has been shown to sensitize CDDP-resistant ESCC cells to CDDP by inhibiting glycolysis and promoting mitochondrial dysfunction. However, the factors that influence the sensitization of CDDP-resistant ESCC cells in vivo are complex and include DNA repair, apoptosis, the tumor microenvironment and tumor stem cells. In the future, it will be necessary to explore and further identify the TPL sensitization pathway.

In conclusion, the present study revealed that TPL may reduce the development of CDDP-resistant ESCC cells in vitro and in vivo. The CDDP-sensitizing effect of TPL could be due to the inhibition of the glycolytic pathway and the induction of mitochondrial impairment in ESCC cells, which impairs the proliferation, migration, invasion and EMT processes of CDDP-resistant ESCC cells and promotes apoptosis to inhibit ESCC tumor growth (Fig. 8). The findings of the present study may serve as a theoretical foundation for the future development of CDDP-sensitizing medicines. For energy metabolism, it is necessary to further observe the changes in ESCC cell proliferation and the tumor microenvironment under different conditions of chemotherapeutic drugs and TPL, as well as to analyze the energy metabolism of ESCC cells under different conditions, in order to further identify the chemonsensitizing mechanism of TPL. Furthermore, in the future, it is necessary to optimize the concentration of TPL and apply it in clinical trials, to conduct more and longer-term follow-up investigations, to analyze the effect of TPL on the prognosis of patients with ESCC treated with radiotherapy and to assess the practical value of its supportive clinical application. Due to their multi-component nature, herbal mixtures can act on multiple pathways and targets in the human body, making them more conducive to improving chemotherapeutic efficacy while reducing toxic side effects. However, the specific clinical effects of ATL-I are still unclear and, in the future, it will be necessary to further improve the toxicity testing and clinical trials and to use gene editing technology combined with transcriptomics, epigenetics and other methods to explore the transcriptional regulation mechanism of ATL-I metabolism, transport, immunity and other related genes.

Figure 8. Diagram of the action mechanism. TPL inhibits glycolysis and causes mitochondrial dysfunction (elevated ROS, decreased mitochondrial membrane potential and ATP and Cytc release), which further inhibits ESCC cell proliferation, migration, invasion and EMT, as well as promotes apoptosis (elevated caspase-9 and caspase-3), thereby inhibiting ESCC cell resistance to CDDP. TPL, triptolide; ROS, reactive oxygen species; Cytc, cytochrome c; ESCC, esophageal squamous cell carcinoma; EMT, epithelial-mesenchymal transition; MMP, mitochondrial membrane potential; CDDP, cis-diamminedichloro-platinum.

TPL may be used in the future, in conjunction with other herbal components, to establish efficient biological screening and formulation research based on herbal components, as well as clinical applications and industrial needs to fully explore the essence of traditional Chinese medicine.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

KL and JiaL conceived and designed the research, conducted experiments and analyzed data as well as drafting and revising the manuscript critically for important intellectual content and confirmed the authenticity of all the raw data. TM, NW and JuL contributed to the acquisition, analysis and interpretation of data and provided substantial intellectual input during the drafting and revision of the manuscript. MQ, LD and JinL participated in the conception and design of the present study and played a key role in data interpretation and manuscript preparation. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by Department of Thoracic Surgery, The First Affiliated Hospital of Zhengzhou University (approval no. 20220096).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References