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. 2020 Oct 14;37(2):128–135. doi: 10.1002/kjm2.12306

Decreased drug resistance of bladder cancer using phytochemicals treatment

Chun‐Jung Cho 1,2, Cheng‐Ping Yu 1, Chia‐Lun Wu 2, Jar‐Yi Ho 1,3, Ching‐Wei Yang 4,✉, Dah‐Shyong Yu 2,✉
PMCID: PMC11896564  PMID: 33280258

Abstract

The aim of the study is to investigate the ability of phytochemicals to overcome the multiple drug resistance (MDR) of bladder cancer. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxic sensitivity of T24‐GCB cells, a GCB resistant cell line, to different phytochemicals, including capsaicin, quercetin, curcumin, and resveratrol, and their combination with gemcitabine. Western blot analysis was used to detect the expression of membranous ABCC2 and metabolic proteins, DCK, TK1, and TK2 in tumor cells. Animal models were used to confirm the treatment efficacy of phytochemicals in combination with gemcitabine to bladder cancer. The observed/expected ratio of cytotoxicity analysis revealed that capsaicin has synergistic effect with gemcitabine to T24‐GCB cells in a dose‐dependent pattern. Quercetin, curcumin, and resveratrol have additive effect with gemcitabine to T24‐GCB cells. Capsaicin and quercetin alone and combination with gemcitabine decreased the expression of ABCC2 and DCK and TKs, in T24‐GCB cells. On the contrary, resveratrol and curcumin alone and combination with gemcitabine increased the expression of ABCC2 but decreased cytoplasmic kinases simultaneously. In xenografted subcutaneous tumor model on nude mice, combination treatment of capsaicin and gemcitabine demonstrated the highest tumor suppression effect when compared to capsaicin or gemcitabine treatment alone. The MDR of bladder cancer is closely related to membranous ABCC2, cytoplasmic DCK, and TKs expression. Capsaicin owns the strongest synergistic cytotoxic effect of gemcitabine to T24‐GCB cells. This combination regimen may provide as an adjunctive treatment for overcoming MDR in bladder cancer.

Keywords: ABCC2 protein, bladder cancer, capsaicin, gemcitabine resistance, phytochemicals

1. INTRODUCTION

Bladder cancer is one of the top 10 cancers. 1 , 2 About 70% of the urinary bladder urothelial cell carcinoma (UCC) is diagnosed initially as well‐differentiated superficial lesions and the rest correspond to highly invasive, poorly differentiated carcinoma. Although patients with superficial tumor initially responded successfully to transurethral resection and adjuvant treatment with intravesical chemotherapy or immune therapy, these patients are at high risk (−60%) of developing recurrent superficial bladder tumor. Of these, 10% to 40% patients progress to invasive metastatic disease and are therefore potentially lethal. 3 , 4

Although bladder cancer is considered to be responsive to the chemotherapy regiments, multiple drug resistance (MDR) is the most common cause of treatment failure and obstacle of intravesical chemotherapy (mitomycin C and gemcitabine) and cisplatin‐based systemic chemotherapy clinically, especially for muscle invasive bladder cancer. 5 Therefore, it is urgent to develop novel treatment strategies that have fewer side effects and are more effective than the currently used therapeutic regimens for bladder cancer.

Phytochemicals are plant‐derived substances that can affect disease development and have been used for the prevention of chronic and degenerative diseases. 6 Targeted therapy is a thriving area of cancer research, which is rapidly developing. As more novel targets will be identified by tumor DNA sequencing and other techniques, as more novel drugs may be developed to inhibit them. Phytochemicals and herbal mixtures act multi‐specifically, that is, they attack multiple targets at the same time. Phytochemicals are considered as therapeutic agents in downregulating the MDR in cancers. 7 In bladder cancer, curcumin can inhibit cell proliferation and reduce migratory and invasive ability via downregulating β‐catenin expression and reversing epithelial‐mesenchymal transition. 8 Apigenin can increase reactive oxygen species (ROS) levels and deplete glutathione (GSH) in bladder cancer. 9 Resveratrol can suppress cell proliferation and induce apoptosis through STAT3 signaling pathway and tumor growth in xenograft model in bladder cancer. 10 Hence, we suppose that phytochemicals could overcome the MDR occurrence of UCC and enhance the cytotoxicity of anticancer drugs.

In our previous studies, the gemcitabine resistant UCC cell line, T24‐GCB, had been established. High expression of ATP binding cassette subfamily C member 2 (ABCC2) mRNA and protein was found in T24‐GCB cells but decreased metabolism proteins of deoxycytidine kinase (DCK), thymidine kinase 1 (TK1), and thymidine kinase 2 (TK2) were seen. In the subsequent study, we further evaluated the cytotoxic enhancement of various phytochemicals on gemcitabine in T24‐GCB cells from both in vitro and xenografted animal tumor model and explored the related mechanisms.

2. MATERIALS AND METHODS

2.1. Establishment of gemcitabine resistant UCC cell line‐T24‐GCB

The gemcitabine resistant UCC cell line, T24‐GCB, was obtained by long‐term culture of T24 cells in progressive escalating concentration of gemcitabine for couple of weeks till the establishment of stable clones. The native T24 and T24‐GCB cells were cultured in RPMI‐1640 medium (Thermo Scientific HyClone, Logan, Utah), supplemented with 10% fetal bovine serum and antibiotics penicillin‐streptomycin. The half of inhibitory concentration (IC) of gemcitabine for native T24 and T24‐GCB were 5.61 ± 1.41 and 105.8 ± 42.7 nM individually.

2.2. MTT cytotoxic assay of various phytochemicals alone and gemcitabine combinations

Cellular chemo‐sensitivity was assayed using a modified MTT assay to determine cell viability in vitro. 11 In brief, T24 and T24‐GCB (1500 cells/well) in 100 μL culture medium were seeded into 96‐well microplates and incubated for 16 hours at 37°C. Cells were treated by phytochemicals (Sigma) alone or cotreated with combination of gemcitabine and various ICs of phytochemicals for 72 hours, respectively. Before the cytotoxic assay, 100 μL of MTT (1 mg/mL in PBS) was added to each well and allowed to react for 3 hours. Then the blue formazan crystals formed was dissolved in 100 μL of DMSO and the optical density was determined by absorbance spectrometry at 560 nm using a microplate reader (Thermo Fisher Scientific, Waltham, US). Three separate experiments with triplicate runs in each were performed to obtain mean cell viability. The drug inhibition concentrations (ICs) were determined using the computer software Calcusyn 1.1 (Biosoft, Cambridge, UK). For each combined effect of growth inhibition or fraction affected (fa), a combination index (CI) is generated. The effects of the combination are then transformed and displayed as fa‐CI plots. CI < 1, = 1, and > 1 indicate synergism, additive, and antagonism, respectively.

2.3. Western blot detection of proteins changes

The underlying mechanisms of ABCC2 for drug resistance in cell membrane and DCK, TK1, and TK2 for drug metabolism in cytoplasm were evaluated after phytochemicals combination with gemcitabine treatment on T24‐GCB cells. Briefly, 106 T24‐GCB cells after phytochemicals combination with gemcitabine or phytochemicals and gemcitabine alone treatment were trypsinized and washed with PBS twice. Cells were suspended in 100 μL of RIPA buffer (Millipore) contained with cocktail protease inhibitor (Roche, Switzerland). 50 μg protein was electrophoresed for 2 hours in 10% SDS‐polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (PVDF, Millipore) by electro blotter with 100 voltages for 1 hour at 4°C. Antibodies ABCC2 (Cell signaling Technology), DCK (Gene Tex Inc), TK1 (Gene Tex Inc), TK2 (Abcam, UK) were diluted in TBST containing 5% nonfat milk and membranes were incubated for 1 hour with gentle agitation. The blots were washed for three times with TBST and incubated with goat anti‐rabbit antibody conjugated to horseradish peroxidase (Cell signaling Technology) for 1 hour. After three successive washes with TBST (tris‐buffered saline (TBS) and Tween20), western blotting chemiluminescence reagent (Bio‐Rad, California) was used for protein detection. The membrane was pictured and analyzed by UVP (LLC).

2.4. Growth inhibition of xenografted tumors by gemcitabine and capsaicin combination

The animal study received institutional animal care and use committee approval (No: IACUC‐17‐105), 8‐weeks‐old female BALB/c nude mice (NALC, ROC) were injected with 0.1 mL T24‐GCB cells (107 cells/ml in 1:1 PBS/FBS) over the left back subcutaneously. One‐week later, four study groups mice: vehicle, gemcitabine (1 mg/kg), capsaicin (10 mg/kg), and combination treatment of gemcitabine with capsaicin, were received consecutively 6 shots (200 μL) of reagents through intraperitoneal injection individually within 2 weeks. The subcutaneous tumors of each group were measured daily with vernier calipers for the long and short dimensions after 6 days of cell injections. After 36 days the mice were euthanized by CO2 inflation into cage with flow rate of 3 L/min till 1 minute after mouse breathing stops and the primary tumors were excised, weighed, and preserved for further analyzes. Tumor volume was calculated as follows: length × width 2 × 1/2. Statistical analysis was performed in SPSS version 24.0 (IBM, Corp.).

2.5. Hematoxylin and eosin stain of tumors

Three micrometer thick sections were prepared from paraffin‐embedded tissue blocks and stained by Hematoxylin and eosin (H&E) method. The sections were deparaffinized and processed as follows: tissue slides were incubated at 75°C oven for 10 minutes, rinsed by xylene 3 minutes three times, 100% ethanol 3 minutes three times, 95% ethanol 3 minutes, 75% ethanol 3 minutes, 50% ethanol 3 minutes, running tap water 5 minutes, and stained with Mayer hematoxylin solution 5 minutes. Slides were rinsed in running cold tap water 5 minutes, 95% acid ethanol 3 seconds 10 times, and stained in eosin Y solution 30 seconds and then dehydrated in 50% ethanol 3 minutes, 75% ethanol minutes, 95% ethanol 3 minutes, 100% ethanol 3 minutes three times, xylene 3 minutes three times for clearing. The slides were evaluated under standard light microscope.

2.5.1. Statistical analysis

Cytotoxicity and protein changes are presented as the mean ± SD. Statistical analysis was performed in SPSS version 24.0 (IBM, Corp.).

3. RESULTS

3.1. Cytotoxic effect of phytochemicals on tumor cells

Six phytochemical compounds (6‐gingerol, apigenin, capsaicin, curcumin, quercetin, and resveratrol) were tested in a serial range of concentrations. The MTT assay revealed that all of these phytochemicals, in addition to 6‐gingerol, own direct cytotoxic effect to both tumor cell lines, T24 and T24‐GCB, with IC50 raged from 3 to 30 μM. Among them, capsaicin had selective cytotoxic effect on native T24 and T24‐GCB cells (Figure 1). T24‐GCB cells were 2‐fold more sensitive to capsaicin treatment than T24 cells in IC50 (40 vs 80 μM).

FIGURE 1.

FIGURE 1

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MTT cytotoxic assay of phytochemicals alone on T24 and T24‐GCB cells in 72 hours

3.2. Cytotoxic enhancement of phytochemical‐gemcitabine combination on T24‐GCB cells

The combined effects of gemcitabine with four selected potential compounds (capsaicin, curcumin, quercetin, and resveratrol) were subsequently analyzed by using MTT assay in T24‐GCB cells. All of them showed enhanced combination cytotoxicity to T24‐GCB cells than gemcitabine treatment alone (Figure 2). In low dose of phytochemical‐gemcitabine combination treatment, capsaicin, quercetin, and resveratrol have increased cytotoxicity to T24‐GCB cells. But no escalating difference in cytotoxicity was seen in high‐dose phytochemical‐gemcitabine combination treatment in addition to capsaicin.

FIGURE 2.

FIGURE 2

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Phytochemical combination with GCB on cell viability for T24‐GCB cells in lower and higher dosages

3.3. Observed/expected ratio of combined phytochemicals and gemcitabine

When observed/expected (O/E) ratio is less than 1.0, it indicates selected compound has synergistic effect on gemcitabine cytotoxicity. On the contrary, when O/E ratio is higher than 1.0, it indicates selected compound has additive effect on gemcitabine cytotoxicity. As shown in Figure 3, capsaicin has synergistic efficacy while curcumin, quercetin, and resveratrol have additive efficacy only and no dose‐dependent relationship was seen among these phytochemicals in enhancing gemcitabine cytotoxicity.

FIGURE 3.

FIGURE 3

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Observed/expected (O/E) cytotoxicity ratio of phytochemicals combination with gemcitabine in T24‐GCB cells according to levels of dosage. (O/E < 1, synergistic effect; O/E > 1, additive effect) (*P < .05)

3.4. MDR pathway changes in tumor cells

Capsaicin and quercetin, either alone or combined with gemcitabine, reduced the ABCC2 expression (Figure 4) while resveratrol and curcumin increased the expression of ABCC2 (data not shown). There were simultaneous decreased expression of cytoplasmic DCK and TKs when T24‐GCB cells were treated by capsaicin, either treatment alone or combined with gemcitabine (Figure 4). Meanwhile, the expression of DCK and TKs also decreased in resveratrol and curcumin treatment (data not shown).

FIGURE 4.

FIGURE 4

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Western blotting of MDR membranous protein ABCC2 and drug metabolism protein expression changes in phytochemicals combination gemcitabine treatment for T24 and T24‐GCB cells. MDR, multiple drug resistance

3.5. Inhibition of xenografted tumor growth

After six consecutive treatments within 12 days, gemcitabine or capsaicin alone demonstrated marginal tumor suppression effect till 36‐day follow‐up end point while their combination treatment had significant inhibition of tumor growth (P = .0049) (Figure 5A,B). Nearly 60% of tumor growth was suppressed by gemcitabine‐capsaicin combination treatment when compared to control mice. Only 10% to 15% of tumor growth was inhibited under gemcitabine or capsaicin treatment alone. Increased lymphocytic infiltration around tumor lesion was observed in capsaicin or gemcitabine‐capsaicin combination treated mice (Figure 5C).

FIGURE 5.

FIGURE 5

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A, Subcutaneous xenografted T24 tumors over the left back in four groups of nude mice. B, Tumor growth volume monitoring in four groups of mice received gemcitabine, capsaicin treatment or their combination (arrow indicates reagent injection) (N = 7). C, Histological examination of four groups of T24‐GCB bearing nude mice (arrow indicates lymphocytic infiltration around the tumor) (H&E x 400). H&E, Hematoxylin and eosin

4. DISCUSSION

Natural phytochemicals were used in cancer prevention and therapy in traditional medicine due to their safety, lack of side effects and their bioavailability, from a wide range of natural sources. Their role in human health and disease is a subject of research. The re‐emergence of phytochemicals in cancer prevention and treatment is demonstrated by the increased number of publications focused on a better comprehension of their biological function and the complex beneficial properties in human health. These were assessed at a cellular, molecular or genomic level using a wide range of cell lines or animal models. 12 , 13 Based on this, phytochemicals were proved to be involved in wide range of key mechanisms in conducting chemoprevention or chemotherapy of cancer. 14 These natural agents are currently exploited for the development of therapeutic strategies alone or in tandem with conventional treatments for cancer in differentiation progression of tumor activation, angiogenesis, and metastasis.

These types of products are classified based on the starting point of their biosynthesis. Phenolics and nitrogen containing compounds are two major classes of phytochemicals in using. Curcumin belongs to phenolic acids subclass, apigenin and quercetin belong to flavonoids subclass, and resveratrol belongs to stilbenes subclass in phenolics (Table 1). Capsaicin and 6‐gingerol belong to nitrogen containing compounds subclass.

TABLE 1.

The studied phytochemicals and their reported structures, involved cancers and related mechanisms

Phytochemicals main classes Subclass Individual phytochemicals Component Chemical structure MW (kDa) Targets and mechanisms Reported cancers References
Phenolics Phenolic acids Curcumin Curcuninoid phytopolyphenols C21H20O6 368.38 ROS, cyclooxygenase(COX) protein kinase C, mitochondria, immune, ABCG2, MDR1, PI3K, p53, apoptosis, autophagy, NF‐kB Breast, head, gastric, lung, colon, prostate ( 12 , 13 , 14 , 17 )
Flavonoids Apigenin Flavonoids C15H10O5 207.24 CYP2C9, bcl‐2, apoptosis, autophagy – ( 15 )
Quercetin Polyphenol C15H10O7 302.236 ROS, free radical, protein kinase (mitochondria), and enzymes – ( 16 , 17 )
Stilbenes Resveratrol Stilbenoid, polyphenol, phytoalexn C14H12O3 228.25 ROS, free radical, mitochondria, and enzymes kinase – ( 18 , 19 , 20 , 23 )
Coumarin – – – – – – –
Tannins – – – – – – –
Nitrogen containing compounds – Capsaicin Capsicum, vanilloids, capsaicinoids C18H27NO3 305.42 TRPV1 receptor, ion channel, COX, NF‐kB, autophagy, apoptosis, PI3K/AKT/mTOR, cell cycle arrest, ROS, FOXO3 NPC, breast, lung, ovary, pancreas, bladder ( 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 )
6‐gingerol – C17H26O4 294.38 – – ( 29 )
Alkaloids – – – – – – – –
Carotenoids – – – – – – – –
Organosulfur compounds – – – – – – – –
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Abbreviation: ROS, reactive oxygen species.

Phenolics (curcumin, apigenin, quercetin, and resveratrol) can inhibit cellular proliferation and inflammation through antioxidant ROS, COX, free radical, protein kinases, and mitochondria interaction according to previous reports with studies in various cancers, including cancer of breast, lung, retinoblastoma, head and neck, stomach, colon, melanoma and prostate. 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 The related pathways of cytotoxicity in phenolics have autophagy, apoptosis activation, STAT3, VEGF, PI3K, p53, bcl‐2, NF‐kB, VEGFR, glycolysis and immune response et al (Table 1).

Nitrogen containing compound (capsaicin and 6‐gingerol) have well known effect on ion channel for TRPV1 receptor, especially on neuron cells. 24 Other studies also have revealed their associated downstream effect on COX, NF‐kB, autophagy, apoptosis, pI3K/AKT/mTOR, FOXO3, p38, JNK, MAPK, ROS, Ca2+‐mediated mitochondrial signal pathways, immune cross talk and cell cycle arrest at G0/G1 in various cancers, including cancers of ovary, breast, lung, pancreas, osteosarcoma, nasopharynx, kidney, and urinary bladder (Table 1). 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32

MDR is a major obstacle in clinical patients with invasive bladder cancer during or after systemic chemotherapy and the outcome usually is lethal. Several mechanisms have implicated in the development of MDR in bladder cancer, including extrusion of the drug by cell membrane pumps, associated with p‐glycoprotein and MDR‐associated protein, increased DNA damage repair, associated with topoisomerase II, p53, and VEGF, suppression of drug‐induced apoptosis and regulation of cancer cell growth. 33 ABC family transporters, including p‐glycoprotein and multidrug resistance‐associated protein (MRP), are actively involved in the MDR of bladder cancer. Among them, ABCB1 and ABCG2 are related with the tumor progression, invasion, and metastasis, and ABCC3 linked to cell proliferation, drug resistance and poor overall survival in gemcitabine and mitomycin C resistant bladder cancer had been reported. 34 Liu et al have reported that both mRNA and protein levels of ABCC3 were significantly higher in urinary bladder cancer tissues than normal tissues. Immunochemistry evaluation of ABCC3 expression showed that high expression of ABCC3 had a positive correlation with tumor size, advanced tumor node metastasis stage, and malignant histology. ABCC3‐knock down cells showed a significant decrease in cell growth and drug resistance. 35 Ojha et al found that gemcitabine and mitomycin C treatment on UCC cells can increase autophagy process via IFN‐γ mediated JAK2 and STAT3 pathway. Combined treatment with inhibitors of JAK2 and autophagy led to inhibition in cell growth, reduced the levels of inflammatory cytokines and decreased the resistance gene expression. 36

Phytochemicals are one of multiple choices for overcoming the MDR of cancer cells. However, which phytochemicals will be able to modulate resistance to chemotherapy in bladder cancer and the possible undertaken mechanisms remains poorly understood. Amantini et al reported that capsaicin triggers autophagic cell survival, which drives epithelial‐mesenchymal transition and chemoresistance in bladder cancer cells in a Hedgehog‐dependent manner. 37 We have established gemcitabine resistance UCC cell line and selected potential effective phytochemicals from phenolics (curcumin, apigenin, quercetin, and resveratrol) and nitrogen containing compound (capsaicin and 6‐gingerol) in this study. The experimental studies have first shown that capsaicin inhibits the growth of gemcitabine resistance bladder cancer cell by inhibiting proliferation in vitro and in vivo though down regulation of ABCC2 expression and cytoplasmic DCK and TKs in a synergistic and dose‐dependent pattern (Figure 6) It has been reported that ABC transporters mediates the major efflux routes of gemcitabine and its metabolites and may contribute to gemcitabine resistance. 38 , 39 , 40 These results indicate that capsaicin can increase intracellular retention of gemcitabine via inhibition of ABCC2 activity in cell membrane.

FIGURE 6.

FIGURE 6

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The scheme demonstrates that capsaicin has been shown to increased cytotoxicity of gemcitabine to UCC tumor cells through decreasing ABC proteins activity in cell membrane with increasing intracellular retention of gemcitabine. UCC, urothelial cell carcinoma

On the contrary, resveratrol and curcumin conduct the cytotoxic enhancement of gemcitabine to bladder cancer cells in an additive pattern through down regulation of cytoplasmic DCK and TKs only. Both of them did not decrease the expression of ABCC2. The role of mitochondria activity and cell cycle changes for the gemcitabine resistant UCC cells and their changes after phenolics or nitrogen containing compounds treatment will be further elucidated in the future.

In conclusion, the gemcitabine resistance of bladder cancer is closely related to increased membranous ABCC2 expression and capsaicin owns the strongest synergistic cytotoxic effect to T24‐GCB cells. The combination strategy of capsaicin and gemcitabine may provide as an alternative treatment for modulation of MDR in chemoresistant bladder cancer.

CONFLICT OF INTEREST

The authors declare no potential conflict of interest.

ACKNOWLEDGMENTS

This study was supported by grants from the Ministry of Science and Technology, Republic of China (MOST‐104‐2314‐B‐016‐040‐MY3, MOST 106‐2320‐B‐016 ‐013 ‐MY3), Tri‐Service General Hospital (TSGH‐C106‐045, TSGH‐D‐109093), National Defense Medical Center (MAB‐109‐064) and Cheng Hsin general hospital (CH‐NDMC‐105‐7, ‐106‐06).

Cho C‐J, Yu C‐P, Wu C‐L, Ho J‐Y, Yang C‐W, Yu D‐S. Decreased drug resistance of bladder cancer using phytochemicals treatment. Kaohsiung J Med Sci. 2021;37:128–135. 10.1002/kjm2.12306

Funding information Ministry of Science and Technology, Republic of China, Grant/Award Numbers: MOST‐104‐2314‐B‐016‐040‐MY3, MOST‐106‐2320‐B‐016 ‐013 ‐MY3; Tri‐Service General Hospital, Grant/Award Numbers: TSGH‐C106‐045, TSGH‐D‐109093; National Defense Medical Center, Grant/Award Number: MAB‐109‐064; Cheng Hsin general hospital, Grant/Award Numbers: CH‐NDMC‐105‐7, ‐106‐06

Contributor Information

Ching‐Wei Yang, Email: chingwei1002@gmail.com.

Dah‐Shyong Yu, Email: yuds45@gmail.com.

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Articles from The Kaohsiung Journal of Medical Sciences are provided here courtesy of Kaohsiung Medical University and John Wiley & Sons Australia, Ltd

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