Triptolide enhances anti-cancer activities of irinotecan in human colon cancer HT-29 cells in vitro and HT-29 cell xenograft tumor model in nude mice

Abstract

Irinotecan (IRI), a clinically used anticancer drug, is effective against various solid tumors, including colorectal cancer, but its use is limited by side effects. Triptolide (TP), an alkaloid from Tripterygium wilfordii, has been used to treat malignancies in China, though its combined effects with IRI on colon cancer cells are not well-documented. In vitro, human colon cancer HT-29 cells were treated with IRI, TP, or both combinations. Results showed that the combination of IRI and TP significantly decreased viable cell numbers more than IRI or TP alone. IRI combined with TP also led to higher Bax and lower Bcl-2 levels, as well as increased cleaved caspase-8, -9, and -3. These findings suggest that TP enhances apoptosis in HT-29 cells and may potentiate the anticancer effects of IRI by disrupting the balance of pro- and anti-apoptotic proteins, which plays a crucial role in controlling tumor cell survival. In vivo, HT-29 cell-xenograft nude mice were treated with IRI, TP, or both combinations for 30 days. Tumor volume and body weight were measured every 2 days, and liver and kidney functions (ALT, AST, CREA, GGT) were assessed. H&E staining of tissues revealed no significant toxicity in the heart, lungs, liver, kidneys, spleen, or small intestine, suggesting that the combination therapy does not induce major organ damage. Immunohistochemical (IHC) analysis showed that IRI combined with TP resulted in higher expression of cleaved-caspase-3, -8, and -9 compared to IRI or TP alone, indicating enhanced tumor cell apoptosis. These results suggest that TP enhances IRI’s anti-cancer effects by promoting apoptosis in colon cancer cells. TP may thus serve as a potential enhancer for IRI in future colon cancer treatments, offering a novel strategy to improve therapeutic outcomes, enhance drug efficacy, and minimize side effects.

Introduction

Colorectal cancer (CRC), one of the most common gastrointestinal malignancies, is associated with high morbidity and mortality worldwide. ¹ In fact, it ranks second in cancer-related mortality globally. ² CRC is the third leading cause of cancer-related deaths in the USA ³ and accounts for 10% of worldwide cancer deaths. ⁴ In Taiwan, the incidence of CRC has been increasing in the past years. According to the 2025 reports from the Ministry of Health and Welfare, CRC is the third most common cancer in Taiwan, accounting for 29.9 deaths per 100 000 individuals annually. ⁵ CRC severity is categorized into stages 0, I, II, III, and IV. Surgery is generally used for stages 0 to III, whereas for stage IV and recurrent colon cancer, surgery is performed in combination with chemotherapy. ⁶ However, cure rates are still unsatisfactory due to side effects. Therefore, finding available compounds from natural products for CRC patients is urgently needed.

Irinotecan (IRI), a hydrophilic compound and 1 of the clinical drugs for cancer patients, acts as an inhibitor of topoisomerase І, ⁷ targeting the S and G2 phases of the cell cycle. ⁷ IRI was obtained from the Chinese tree named Camptotheca acuminata (NyssaceaeBotn). In the United States, IRI has been used as an anticancer drug since 1996, particularly for the treatment of metastatic colorectal cancer.^8,9 IRI was combined with 5-fluorouracil or leucovorin as a first- or second-line therapeutic drug for CRC patients. ¹⁰ Although there have been improvements in cancer chemotherapy, these treatments are still not fully satisfactory due to adverse effects associated with irinotecan, such as severe diarrhea, neutropenia, nausea, and fatigue, which can limit its clinical efficacy.^11,12

Numerous studies have demonstrated that extracts from common fruits, vegetables, and nutritional herbs exhibited growth-inhibitory effects on human cancer models in vitro and in vivo. ¹³ Triptolide (TP), an alkaloid, was purified from traditional Chinese herbs such as Tripterygium wilfordii Hook. f. (Celastraceae). It has been used as a treatment for inflammatory, autoimmune, and malignant diseases in the Chinese population since ancient times. ¹⁴ TP has been reported to exhibit anti-tumor activity in many human cancer cell types,^15,16 such as leukemia, ¹⁷ breast cancer, ¹⁸ cholangiocarcinoma, ¹⁹ gastric cancer, ²⁰ ovarian cancer, ²¹ pancreatic cancer, ²¹ prostate cancer, ²² lung cancer, ²³ and colon cancer. ²⁴ Although numerous studies have shown that TP possesses anticancer activities in vitro and in vivo, there is no available information demonstrating the efficacy of TP combined with IRI against human colon cancer. Therefore, we present the synergistic effects of TP with IRI in colon cancer HT-29 cells in vitro and HT-29 cell-xenograft bearing mice in vivo.

Materials and Methods

Chemicals, Cell Culture Medium, and Antibodies

Dimethyl sulfoxide (DMSO), irinotecan (IRI), and triptolide (TP) were obtained from Sigma Chemical Co. (St. Louis, Missouri, USA). IRI and TP were dissolved in DMSO throughout the whole experiment. Roswell Park Memorial Institute (RPMI) 1640 medium, L-glutamine, penicillin/streptomycin (PS), and heat-inactivated fetal bovine serum (FBS) were purchased from Gibco/Life Technologies (Carlsbad, California, USA). For western blotting: anti-Bax, -Bcl-2, -AIF, -BiP, -XIAP, -caspase-3, -caspase-8, -caspase-9, and HRP-linked antibody were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Primary antibodies against GADD153 were obtained from Sigma Chemical Co. (St. Louis, MO, USA), and those against EndoG were purchased from Millipore Sigma (Billerica, MA, USA). The internal control GAPDH was from Gene Tex Inc. (Alton Pkwy Irvine, CA, USA). For tissue immunohistochemistry staining, the primary antibody against EndoG was purchased from Elabscience (Houston, TX, USA), while all other primary antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA), and all primary antibodies were used at a dilution of 1:200.

Culture of HT-29 Human Colon Cancer Cells

HT-29 human colon cancer cell line (ATCC Cat# HTB-38, RRID:CVCL_0320) was purchased from the American Type Culture Collection (Rockville, MD, USA) and cultured in RPMI 1640 medium (cat. no. 11875093; Gibco; Thermo. Fisher Scientific. Inc.) supplemented with 10% FBS and penicillin (100 U/ml)/streptomycin (100 µg/ml) in culture flasks at 37°C in a humidified with 5% CO₂ and 95% air as described previously. ²⁵

Cell Viability Assays

To evaluate the cytotoxic effect, HT-29 cells at the density of 1 × 10⁴ cells were placed in 96-well plates with 3 replicate wells in each treatment, and incubated overnight. The cells were then treated with fresh medium containing various concentrations of IRI (0, 2.5, 5, and 10 μM), TP (0, 1.25, 2.5, 5, 10, and 20 μM), or TP combined with or without IRI (10 μM) for further 48 hours. After treatment, cell viability was determined using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan) according to the manufacturer’s instructions. The absorbance (OD value) was measured at 450 nm using a microplate reader to evaluate the viable cell number as described previously. ²⁶

Measurements of Reactive Oxygen Species (ROS), Intracellular Ca²⁺ and Mitochondrial Membrane Potential (ΔΨ_m)

HT-29 cells at a density of 2 × 10⁵ cells/well were seeded in 12-well plates with triplicate and were treated with 10 μM of IRI, 10 μM of TP, or 10 μM of IRI combined with 10 μM of TP for 48 hours. Cells were harvested and re-suspended in 500 μl of DCFH-DA (10 μM), 500 μl of Fluo-3 AM (2.5 μg/ml), or 500 μl of DiOC₆ (4 μmol/l) for 30 minutes for ROS (H₂O₂) measurement, intracellular Ca²⁺ measurements, or ΔΨ_m levels measurements, respectively. All cells were analyzed for ROS, Ca²⁺, and ΔΨ_m levels by FACSCalibur Flow Cytometer (BD Biosciences, FACSCalibur, San Jose, CA, USA) as described previously. ²⁶

Caspase-3 Activity Assay

HT-29 cells (2 × 10⁵ cells) were seeded in 12-well plates with triplicate and were pretreated with caspase substrate (PhiPhiLux^R-G₁D₂) and treated with 10 μM of IRI, 10 μM of TP or 10 μM of IRI combined with 10 μM of TP for 48 hours. All cells were analyzed for caspase-3 activity by flow cytometry as described previously. ²⁷

Western Blotting Assays

HT-29 cells (1 × 10⁶ cells) were maintained in 10 cm dishes overnight and were treated with 10 μM of IRI, 10 μM of TP, or 10 μM of IRI combined with 10 μM TP for 48 hours. Cells were harvested and then lysed using a PRO-PREP^TM Protein Extraction Solution (iNtRON Biotechnology, Seoul, Korea). The total proteins from each treatment were quantitated by a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Inc.) as previously described. ²⁷ The same amount of denatured protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it was then transferred to an Immobilon P Transfer Membrane (Merck Millipore). Then, the membrane was blocked with 5% skimmed milk in blocking buffer and incubated with the corresponding primary antibodies, including anti-AIF (#4642, RRID:AB_2224542, dilution 1:1000), -Bax (#2772, RRID:AB_10695870, dilution 1:1000), -Bcl-2 (#3498, RRID:AB_1903907, dilution 1:1000), -BiP (#3177, RRID:AB_2119845, dilution 1:1000), -cleaved caspase-3 (#9662, RRID:AB_331439, dilution 1:1000), -caspase-8 (#9746, RRID:AB_2275120, dilution 1:1000), -caspase-9 (#9508, RRID:AB_2068620, dilution 1:1000), anti-EndoG (#AB3639, RRID:AB_11211929, dilution 1:1000), -GADD153 (#G6916, RRID:AB_259948, dilution 1:400), GAPDH (#GTX100118, RRID:AB_1080976, dilution 1:200 000), and -XIAP (#2042, RRID:AB_2214870, dilution 1:1000) overnight at 4°C. After being washed 3 times with Tris-buffered saline-0.1% Tween 20 solution, the membranes were incubated with relevant anti-rabbit IgG, HRP-linked antibody (#7074, RRID:AB_2099233, dilution 1:10 000; horseradish peroxidase-conjugated secondary antibody) at room temperature for 1 hour, followed by using chemiluminescence detection with ECL (Merck Millipore) as previously described. ²⁷

Creating the Colon Cancer HT-29 Cell-Xenograft Bearing Mice and the Treatments of IRI, TP, or IRI Combined With TP

Male 6-week-old nude mice (CAnN.Cg-Foxn1nu/CrlNarl mice, RRID:IMSR_CRL:194) with 20 to 22 g bodyweights were purchased from the National Laboratory Animal Center, Taipei, Taiwan. The mice were subjected to a 12-hour light/dark cycle, with free access to water and standard mice chow, housed under pathogen-free conditions at the Animal Center of China Medical University (Taichung, Taiwan). The animals were randomly divided into 4 groups, as outlined in the flow chart shown in Figure 4A. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) in China Medical University with ID number CMUIACUC-2023-265.

Figure 4.

IRI, TP, and the combination of IRI with TP suppressed HT-29 cell tumor growth. The animal experimental flow chart is shown (A). The body weights of each group animal were recorded per 2 days (B). The tumor volume of control and experimental groups were measured per 2 days (C). The tumors were isolated and present after treatment (D). All are performed as described in Materials and Methods. P-value was shown and P < .05 indicated a significant difference.

A total of 1 × 10⁶ HT-29 cells were inoculated subcutaneously into the right flank on each nude mouse for 7 days. Tumor size was measured by calipers. After the subcutaneous solid tumor reached 100 to 120 mm³, all mice were randomly assigned into four treatment groups (n = 3 per group). All mice were treated with test agents by oral gavage once daily for 30 consecutive days. Group I mice were fed with 0.1% DMSO in 100 μl phosphate-buffered saline (PBS) as the control. Group II mice were fed with 6 mg/kg of IRI in 100 μl PBS. Group III mice were fed with 3.5 mg/kg of TP in 100 μl PBS. Group IV mice were treated with the combination of 6 mg/kg IRI and 3.5 mg/kg TP in 100 μl PBS. Body weight and tumor size were individually measured and recorded every 2 days. Xenograft tumor volume (mm³) was calculated as follows. The formula (V = Length × width² × 0.523) was used to calculate the tumor volume in each mouse as described previously. ²⁸

Quantitation of Alanine Transaminase (ALT), Aspartate Transaminase (AST), Creatinine (CREA), and gamma-Glutamyl Transpeptidase (GGT)

For analysis of CREA, ALT, AST, and GGT in mice, the blood samples were isolated from each mouse during IRI, TP, and IRI combined with TP treatment for 30 days. Isolated blood samples were centrifuged at 2000 rpm for 20 minutes, and collected serum and kept at 4°C for further determination of creatinine (CREA) by using Automated Clinical Chemistry Analysis System, Dimension type RXL Max (Dade Behring Delaware, DE 19714, USA). ALT, AST, and GGT were analyzed according to the guidelines from Axel Biotechnology Inc. (Taichung, Taiwan) as described previously. ²⁹

Histological and Immunohistochemical Assay

After 30-day treatment, all animals’ tumor, lung, heart, spleen, kidney, liver, and small intestine tissues were collected. Hematoxylin and Eosin (H&E) staining was conducted to examine whether IRI, TP, and IRI combined with TP induced the toxicities in animals. Staining was performed by Bio-Check Laboratories Ltd (New Taipei City, Taiwan) as described in Liu et al study. ³⁰ To further examine the apoptosis in tumor tissues, tumors were removed and IHC staining was conducted, measuring apoptosis-associated protein expression following the EMD Millipore’s IHC Select^® kit instructions (EMD Millipore, Billerica, Massachusetts, USA), as described in our previous study. ³¹ After slides were probed with antibodies, all samples were examined and photographed by Nikon microscope at 20× magnification (Nikon ECLIPSE Ti-U, Minato City, Tokyo, Japan). ImageJ software version 1.50 (National Institutes of Health, Bethesda, MD, USA; RRID:SCR_003070) was used to quantify the specific protein in IHC stain as described previously. ²⁸

Statistical Analysis

All data were represented as mean ± standard deviation. In vitro experiment, 1-way ANOVA analysis was used to compare means between control and treatment groups. For the in vivo experiment, the 1-way ANOVA statistical analysis used the GraphPad Prism 7.0 version (San Diego, California, USA, RRID:SCR_002798). Further, the means between control and experiment groups (in vivo study) were compared by Dunnett’s post-hoc test. The P-value less than .05 was defined as a significant difference.

Results

IRI, TP, and IRI Combined With TP Decreased Viable Cell Number in HT-29 Cells

To assess the effects of cytotoxic effects of IRI, TP, or IRI combined with TP on HT-29 cells, we assessed the total viable cells after HT-29 cells were treated with IRI, TP, or IRI combined with TP for 48 hours. Results are presented in Figure 1A and B. Cell viability was not significantly different across concentrations of 2.5 to 10 μM of IRI treatment alone (Figure 1A). The effects of IRI combined with TP on cell viability in HT-29 cells were further examined and the results are presented in Figure 1B. We found that the combination of IRI with TP significantly decreased cell viability compared to the IRI or TP only treatments, and that the result was dose-dependent. Thus, TP could enhance IRI-induced cell cytotoxicity in HT-29 cells.

Figure 1.

IRI, TP, and combination of IRI with TP decreased cell viability in HT-29 cells. Cells (1 × 10⁴ cells/well) were placed in 96-well and were treated with IRI (A), TP, and the combination of IRI with TP (B) as shown in concentrations for 48 hours. After treatment, cells were collected to measure cell viability, as described in Materials and Methods.

*A significant difference between control and test treatments. #1A significant difference between IRI and IRI combined with TP treatments; #2 A significant difference between TP and IRI with TP treatments.

IRI, TP, and IRI Combined With TP Affected ROS, Ca²⁺ and ΔΨ_m Levels in HT-29 Cells

To investigate whether ROS, Ca²⁺, and ΔΨ_m are involved with cell cytotoxicity of IRI combined with TP treatment on HT-29 cells, cells were exposed to IRI, TP, or IRI combined with TP for 48 hours and then collected for assays of ROS and Ca²⁺ production and ΔΨ_m levels. The results are presented in Figure 2. Results indicated that treatment with IRI, TP, or IRI combined with TP increased ROS production 1.5-, 3.2-, and 2.3-fold, respectively (Figure 2A); increased Ca²⁺ production 2.1-, 3.2-, and 4.2-fold, respectively (Figure 2B); and caused fold changes in ΔΨ_m levels of 1.4-, 0.7-, and 0.6- respectively (Figure 2C). Results indicated that co-treatment with TP and IRI induced the highest levels of Ca²⁺ and caused the greatest decrease in ΔΨ_m levels compared to control HT-29 cells. Although the combination treatment did not induce the highest ROS levels among all groups, it still significantly elevated ROS production compared to the control.

Figure 2.

IRI, TP, and the combination of IRI with TP affected ROS, Ca²⁺, ΔΨ_m, and caspase-3 activity in HT-29 cells. Cells (2 × 10⁵ cells/well) were treated with or without IRI, TP, and the combination of IRI with TP as shown in concentrations for 48 hours. After treatment, cells were collected for measuring the production of ROS (A) and Ca²⁺ (B) and ΔΨ_m levels (C) by flow cytometer and calculated the percentage as described in Materials and Methods. Or cells were treated with IRI, TP, and the combination of IRI with TP for 48 hours and then treated with caspase-3 substrate for further measuring the caspase-3 activity (D) as described in Materials and Methods.

*A significant difference between control and test treatments. #1 A significant difference between IRI and IRI combined with TP treatments. #2 A significant difference between TP and IRI with TP treatments.

HT29 cells were incubated with IRI, TP, or IRI combined with TP for 48 hours, and the substrate of caspases-3 (PhiPhiLux^R-G₁D₂) was added to cells. The collected cells were then measured for caspase-3 activity and the results are presented in Figure 2D. Results indicated that IRI combined with TP induced higher caspase-3 activity (2.2-fold) than IRI (1.6-fold) or TP (1.7-fold) treatment alone in HT-29 cells. Thus, the cell cytotoxic effects of the combination of IRI and TP is involved in activating caspases in HT-29 cells.

IRI, TP, and IRI Combined With TP Affected the Apoptosis-Associated Protein Expression in HT-29 Cells

After HT-29 cells were incubated with IRI, TP, or IRI combined with TP for 48 hours, all cells from each treatment were separately collected for western blotting assay, and results are presented in Figure 3. Figure 3A shows that IRI and TP treatment increased Bax (pro-apoptotic protein), AIF, and EndoG; but decreased Bcl-2 (anti-apoptotic protein). However, combination treatment with IRI and TP increased Bax, AIF, and EndoG and decreased Bcl-2 to a greater extent than either IRI or TP treatment alone. Moreover, the IRI and TP combination treatment increased caspase-3, caspase-8 caspase-9, Bip, and GADD153; and decreased XIAP to a greater extent than the IRI and TP treatments alone in HT-29 cells (Figure 3B).

Figure 3.

IRI, TP, and the combination of IRI with TP affected apoptosis-associated protein expression in HT-29 cells. Cells (1 × 10⁶ cells/dish) were treated with or without IRI, TP, and combination of IRI with TP as shown in concentrations for 48 hours. Cells were collected. lysed and quantitated protein concentration to examine apoptosis-associated protein expression by western blotting as described in Materials and Methods. (A): Bax, Bcl-2, AIF, EndoG; (B): caspase-3, caspase-8, and caspase-9, XIAP, BiP, and GADD153. GAPDH was used as an internal control.

IRI, TP, and IRI Combined With TP Affected Body Weights and Colon Tumor Growth in HT-29 Cell- Xenografted Mice

In order to confirm the anti-tumor activity of IRI, TP, and IRI combined with TP in vivo, HT-29 cell subcutaneous xenograft mice were developed. The flow chart of the experiment is presented in Figure 4. Mice were treated with IRI, TP, or IRI combined with TP for 30 days. We recorded body weight every 2 days during treatment and the average body weight from each HT-29 cell-bearing xenograft mouse is presented in Figure 4B. IRI, TP, or IRI combined with TP did not significantly affect body weight. The average of tumor volume during the experiment is presented in Figure 4C. After 30 days of treatment the tumors were isolated and are presented in Figure 4D. IRI and TP separated treatments show inhibited tumor volumes; however, the combination of IRI with TP induced greater inhibition of tumor volume than that of IRI or TP treatment only (Figure 4C). These observations show that IRI combined with TP effectively suppressed the growth of mouse tumors (tumor volume), and that a significant difference occurred after day 12 of treatment (Figure 4C).

IRI, TP, and IRI Combined With TP Treatment Affected Acute or Decreased Toxicity of HT-29 Cell-Bearing Mice

At the experimental endpoint, heart, lung, liver, kidney, small intestine, spleen, and tumor tissues were collected and examined for signs of toxicity using H&E staining to assess the effects of IRI, TP, and IRI combined with TP. These tissues underwent a macroscopic examination based on histopathological observations for monitoring tissue integrity and injuries that are known signs of toxicity such as cell degeneration (the deterioration of cell structure and function), cell necrosis and apoptosis.^32,33 These results are shown in Figure 5. Based on these observations, no significant pathological differences (toxicities) developed in the heart, lung, liver, kidney, small intestines, and spleen organs among the 4 treatment groups in nude mice.

Figure 5.

The examinations of IRI, TP, and the combination of IRI with TP induced toxicity from xenograft HT-29 cell-bearing mice. After treatment, heart, lung, liver, kidney, spleen, small intestines, and tumors were isolated from each mouse and H&E staining were performed for further examination of the pathology photograph under the microscope with 100 times magnification as described in Materials and Methods.

IRI, TP, and IRI Combined With TP Affected the Levels of Alanine Transaminase (ALT), Aspartate Transaminase (AST), Creatinine (CREA), and gamma-Glutamyl Transpeptidase (GGT) In Vivo

Blood samples were isolated from individual mice and measured for the levels of AST, ALT, CREA, and GGT, which are presented in Figure 6. The observed levels of ALT, AST, CREA, and GGT were within the normal range for mouse models. ³⁴ The AST assay showed lower AST levels in all the test groups. However, these results showed no significant differences in the treatments of IRI, TP, or IRI combined with TP compared to the control.

Figure 6.

The effects of IRI, TP, and IRI combined with TP on the levels of alanine transaminase (ALT), aspartate transaminase (AST), creatinine (CREA), and gamma-glutamyl transpeptidase (GGT). The nude mice serum from each group was collected on day 30 for ALT (A) and AST (B), CREA (C), and GGT (D) as described in Materials and Methods. ns, no significant difference between the control and treated group.

IRI, TP, and IRI Combined With TP Affected Anti-Apoptosis and Pro-apoptosis Factors in HT-29 Cell-Bearing Mice

Individual tumors were further examined for protein expression validation by using IHC staining and the results were presented in Figure 7. The expression of Bax and Bak, 2 pro-apoptosis proteins, as well as AIF and EndoG, all significantly increased in response to treatments relative to the control (Figure 7A and B). Conversely, the expression of Bcl-2, Mcl-1, XIAP, and c-FLIP, which are all anti-apoptotic proteins, decreased, with the IRI combined with TP treatment having the greatest decrease when compared to the IRI or TP treatments alone (Figure 7C and D). We showed that the IRI, TP, and combination IRI with TP treatments induced cytotoxic effects in HT-29 cells in vitro (Figure 1). The results from IHC staining indicate that IRI, TP, and the combination of IRI with TP increased the protein expression of cleaved caspase-3, caspase-8, and caspase-9 in tumor tissues, as shown in Figure 7E and F. Moreover, the combination treatment (IRI with TP) induced the greatest increase in expression of cleaved caspase-3, caspase-8, and caspase-9. Thus, TP may enhance the effects of IRI-induced cell apoptosis by leading to a suppression of tumor growth in vivo.

Figure 7.

IRI, TP, and combination of IRI with TP suppressed apoptosis-associated proteins in tumors. At the end of treatment, tumors were isolated from each mouse and stained by IHC. The IHC staining images of AIF, EndoG, Bax, and Bak (A and B); c-FLIP, XIAP, Bcl-2, and Mcl-1 (C and D); cleaved caspase-3, -8, and -9 (E and F) were examined and photographed by microscope with 20 times magnification and were further quantified as described in Materials and Methods. P-value was shown and P < .05 indicated a significant difference.

Discussion

Globally, colon cancer is a significant contributor to cancer-related morbidity and mortality in the human population. ¹ Currently, the major treatment for human colorectal cancer (CRC) is surgery plus chemotherapy which involves 5-Fluorouracil (5-FU) treatment. Based on statistical reports, the advantages of 5FU for clinical use have been shown, however, the overall response rate in patients for advanced CRC is only about 10% to 15%. ³⁵ Treatment with 5FU in combination with other anti-tumor drugs may lead to improved response rates of 40% to 50%. ³⁶ Side effects of clinically used drugs are also major obstacles to this treatment. In addition, 5FU-resistant cancer has developed in response to CRC treatment. ³⁷ Advancing therapeutic methods should be a focus.

IRI, one of the clinical use drugs in CRC patients, inhibits tumor growth via induced cancer cell apoptosis in human colon cancer cells.^38,39 Currently, the induction of tumor cell death via cell apoptosis is recognized to be one of the best strategies for inhibiting tumor growth and aggressiveness in cancer patients; it is one of the ultimate goals of neoplastic therapy. Because cancer cells can become insensitive to death after being exposed to anticancer drugs, further development of apoptosis-based treatments has been a main focus of attention in cancer research. ⁴⁰

We selected TP for this study because it is a natural product with low cytotoxicity and low induction of side effects. Many reports have shown that TP inhibits tumor cell growth through inhibiting cell proliferation, inducing cell cycle arrest and cell apoptosis.^41-43 Here we selected IRI combined with TP to investigate the anticancer activity of this combination treatment against human colon cancer HT-29 cells in vitro and in vivo.

In vitro HT-29 cell experiments indicate that IRI combined with TP induced higher cell death than IRI or TP treatment alone (Figure 1). Thus, we selected the combination of IRI (10 μM) and TP (10 μM) to effectively inhibit the proliferation of HT-29 cells for further experiments. It is well documented that endoplasmic reticulum (ER) stress and dysfunction in mitochondria both play a critical role in cell apoptosis.^44,45 Thus, in the present study, we found that the combined TP and IRI treatment significantly increased the production of both Ca²⁺ and ROS, and significantly decreased ΔΨ_m levels, to a greater extent than either IRI or TP treatment alone. Agent-induced cell apoptosis through the activities of caspase-8, -9, and -3 has been previously demonstrated, and therefore some anticancer drugs induce cell apoptosis via caspase-dependent pathways.^46,47 Our earlier studies showed TP-induced cell apoptosis in human melanoma cancer cells through a caspase-dependent pathway. ⁴⁸ In this study, the treatment with a combination of IRI and TP induced a higher caspase-3 activity in HT-29 cells than treatment with either IRI or TP alone (Figure 2). We conclude that the combination of IRI and TP may induce cell apoptosis in HT-29 cells through caspase-dependent pathway.

We further set up experiments to find out possible proteins associated with cell apoptosis and to understand the molecular mechanisms that might be involved in cell apoptosis, a western blot assay was used to measure the effects of IRI, TP, or IRI combined with TP treatments on cell apoptosis-associated protein expression in HT-29 cells. The combination of IRI with TP induced higher pro-apoptotic protein such as Bax but lower anti-apoptotic proteins such as Bcl-2 than that of IRI or TP treatment alone (Figure 3A). Furthermore, the combination treatment of IRI with TP increased expression of AIF and EndoG more than that of the IRI or TP treatments alone (Figure 3A). Both AIF and EndoG release from mitochondria and will directly move to the nucleus to induce cell apoptosis without involving caspase-9 and -3. Furthermore, results from Figure 2C also showed IRI combined with TP showed a larger decrease in ΔΨ_m levels than that of IRI or TP treatment alone. Figure 3B showed IRI combined with TP induced higher cleaved caspase-8, -9, and -3 than that of IRI or TP treatment alone. Apoptotic cell death may be caused via FAS ligand binding to the FAS receptor and then activation of caspase-8 to form active caspase-8, which activates executioner caspase-3 for cell apoptosis.^49,50 Therefore, the combination of IRI and TP induced cell apoptosis through caspase-dependent pathways. Alternatively, the disruption of the ratio in Bak/Bcl-2 could reduce the ΔΨ_m for cytochrome c release and activate caspase-9 following active caspase-3/7 for cell apoptosis. ⁵¹ Bak and Bcl-2 belong to the Bcl-2 protein family which is one of the key apoptosis regulators associated with the outer mitochondrial membrane. ⁵² Bcl-2 (anti-apoptotic protein) protects the membrane integrity and keeps cytochrome c in mitochondria, however, it can be disrupted by Bax (pro-apoptotic protein). ⁵² From these observations, TP enhanced IRI-induced apoptotic cell death in HT-29 cells in vitro may be through mitochondria- and caspase-dependent pathways.

To further investigate whether the TP enhancement in IRI-induced cell apoptosis is developed in vivo, thus, HT-29 cells were injected into nude mice until all tumor growth went up to 100 mm². All mice were classified into 4 groups at random and treated with IRI, TP, or IRI combined with TP for 30 days and both their body weights and tumor volumes were recorded for every 2 days during treatment as shown in Figure 4A. We calculated both averages of body weights and tumor volumes from each mouse per group as presented in Figure 4B and C, respectively. Based on the data from Figure 4B, IRI, TP, and IRI combined with TP showed no effect on the body weights of these mice due to no statistical difference among these groups.

At the end of treatment, first, a blood sample was collected, heart, liver, lung, spleen, small intestine, and kidney were removed from individual mice in each group for further experiments. All collected blood samples were further measured for AST, ALT, CREA, and GGT, and results indicated that IRI and TP did not significantly affect the levels of AST, ALT, CREA, and GGT (Figure 6). The normal range of AST, ALT, CREA, and GGT are 30 to 130 U/l, 50 to 350 U/l, 0.2 to 0.5 mg/day, and 3 to 8 U/l, respectively. ⁵³ The isolated heart, lung, liver, kidney, spleen, small intestines, and tumors were further examined by using H&E staining, and the results are shown in Figure 5. These results indicated that IRI, TP, or IRI combined with TP at these doses did not significantly induce cytotoxic effects on these examined tissues. The interesting part is that IRI treated and TP treated individuals significantly decreased tumor volume, but the combination of IRI with TP had a higher decreased tumor volume than that of IRI or TP treatment alone (Figure 4C). These results may suggest that TP-enhanced IRI inhibits tumor growth in nude mice.

For further confirmation of whether IRI combined with TP suppressed tumor growth to a greater extent than IRI or TP alone, we next wanted to investigate whether this involved apoptosis development. We used IHC staining for all isolated tumor tissues and results showed that Bax and Bak (pro-apoptosis proteins; Figure 7A and B are increased but Bcl-2 and Mcl-1 and XIAP (anti-apoptosis proteins; Figure 7C and D) were decreased in IRI or TP treatment. However, IRI combined with TP treatment resulted in a higher promotion or inhibition than that of IRI or TP treatment alone. Anticancer drugs induced cancer cell apoptosis through the increase of pro-apoptotic proteins and the decrease of anti-apoptotic proteins. Furthermore, caspases have been shown to be involved in cell apoptosis,^54,55 thus, in the present studies, IRI combined with TP induced cancer cell apoptosis which through the caspases-dependent pathway.

Results from Figure 7E and F indicated that the combination of IRI with TP has a higher expression of cleaved caspase-3, -8, and -9 than that of IRI or TP treatment alone. These results, consistent with our in vitro data (Figure 3B), support the conclusion that the IRI, TP, and IRI combined with TP induced apoptosis cell death through a caspase-dependent pathway. It was also reported that the up-regulation of the caspase-3-dependent pathway through the induction of cell apoptosis inhibits the growth of human colon cancer cells. ⁵⁶ Our findings are in agreement, showing that in HT-29 cells the down-regulation of the Bcl-2-associated apoptosis regulator/Bcl-2 ratio and the up-regulation of the caspase-9-dependent pathway may promote cell apoptosis. ⁵⁶ The combination of IRI with TP treatment has a higher level of a pro-apoptotic protein (Bak) and a lower level of an anti-apoptotic protein (Bcl-2), and at the same time, cleaved caspase-3, -8, and -9 have induced similar trends in HT-29 cell generated tumor-bearing mice. Taken together, the caspase-related apoptotic pathway may be one of the primary mechanisms for IRI combined with TP to exert its synergistic effect on HT-29 cells. Results from in vitro and in vivo experiments indicated that IRI combined with TP show significant and synergistic effects for reducing cell viability and decreased tumor volumes that may be through the induction of cell apoptosis via mitochondria- and caspase-dependent pathways in HT-29 cells. Other earlier reports have shown that TP exerts a synergistic effect with carboplatin (CBP, a DNA-damaging drug) on the inhibition of cell viability, migration, invasion, and induction of cell apoptosis of human skin cancer A375 and B16 cells. ⁵⁷ In vivo studies also showed that TP and CBP significantly inhibited melanoma tumor progression in nude mice through cell proliferation inhibition and induced cell apoptosis. ⁵⁸ Apoptosis is a pattern of molecular cascades for the removal of the dying cells ⁵⁹ and it is highly controlled by molecular signaling in cells and is closely associated with tissue development, homeostasis, and diseases. ⁶⁰

In conclusion, this work sheds light on TP enhancement of IRI antitumor activity in vitro and in vivo. We offer possible molecular mechanisms for signaling pathways, in particular, via increased cleaved-caspase-3, -8, and -9, increased Bax and Bak, and decreased Bcl-2 and XIAP, which also accompany decreased mitochondria membrane potential for leading to caspase-3 activation or directly to release AIF and EndoG for causing cell apoptosis in HT-29 cells.