Evaluation of Su Fu’ning Lotion’s Inhibitory Effects on Bladder Cancer Cells In Vitro and In Vivo by Intravesical Instillation

Abstract

Background. Bladder cancer is a common malignant tumor with a very high recurrence rate after surgery. Intravesical instillation can help clear up the residual tumor cells after surgery and thereby reduce the recurrence rate. Objective. To establish a bladder tumor transplantation animal model and to evaluate the inhibitory effects of a novel perfusate, Su Fu’ning Lotion (SFN), on bladder tumor. Methods. SFN was compared with several commonly used chemotherapy drugs, including mitomycin (MMC) and pirarubicin (THP) for anticancer effects on the bladder cancer cell lines T24, BTT, and BIU-87 and SFN half inhibitory concentrations (IC₅₀) were determined after 48 hours of treatment. In addition, bladder cancer orthotopic transplantation tumor models were established in BALB/C nude mice and T739 mice, and SFN anticancer effects were assessed in vivo, with normal saline and MMC as negative and positive controls, respectively. Results. SFN, MMC, and THP were all lethal to bladder cancer cells, in vitro, with SFN and THP significantly superior to MMC. IC₅₀ values for SFN were 13.22, 11.22, and 12.5 µg/mL on T24, BTT, and BIU-87 cells, respectively. In vivo, SFN significantly reduced the mouse bladder wet weight and prolonged the animal survival compared with controls (P < .05), suggesting that SFN significantly inhibited T24/BTT cell growth in mice. Conclusion. SFN inhibited the bladder cancer cell proliferation in vitro and in vivo and significantly prolonged the survival of mice with bladder cancer xenografts, indicating that SFN could be used as a perfusate after surgery for removal of residual bladder cancers cells.

Introduction

Urothelial bladder cancer, the 7th most common cancer in men and 17th most common cancer in women worldwide, is more common in developed countries. ¹ Considering the frequency along with its relapsing nature, the bladder cancer requires the highest lifetime treatment costs per patient of all cancers. ²

Intravesical instillation helps to clear up the residual tumor cells left after surgery, and it is one of the most important measures used to prevent postoperative recurrence and reduce the mortality of patients. ³ Indeed, intravesical instillation of pharmorubicin resulted in 50% reduction of the cancer recurrence rate, ⁴ and postoperative intravesical instillation treatments generally showed good therapeutic outcomes. ² However, the drugs used in intravesical instillation could cause chemical cystitis, cystospasm, and other untoward events.^5,6

Sappan wood (Caesalpinia sappan L.) is a traditional Chinese herbal medicine, and its extract has shown promising effects against many bladder cancer cell lines. Brazilin was identified as one of the most active constituents in sappan wood. ⁷ In recent studies, the anti-inflammatory effects of brazilin in macrophages via novel mechanisms involving heme oxygenase-1 (HO-1) ⁸ and inducible nitric oxide synthase (iNOS) gene expression ⁹ were reported. The suppressive effect on iNOS gene expression by brazilin might provide a possible mechanism for its chemopreventive activity in cancer therapy. ⁹ In addition, it has been shown that brazilin protects rat hepatocytes from bromotrichloromethane-induced oxidative injury through HO-1 expression. ¹⁰ Furthermore, brazilin has shown to increase glucose transport through recruitment of glucose transporter type 4 from intracellular pools to the plasma membrane in adipocytes via activation of phosphatidylinositide 3-kinase, ¹¹ inhibit the downstream signaling events of cyclic adenosine monophosphate pathways, ¹² and suppress the hepatic gluconeogenesis by increasing fructose-2,6-bisphosphate levels in hepatocytes. ¹³ Brazilin has been reported to have a wide range of biological activities, which includes potent antibacterial activity against antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, multidrug-resistant Burkholderia cepacia, and other bacteria, ¹⁴ inducing vasorelaxation as well as release of alpha-1-adrenergic receptor agonists in phenylephrine-precontracted rat aortic rings,^15,16 antiplatelet activity, ¹⁷ inducing cellular immune response, ¹⁸ decreasing suppressor cell activity, ¹⁹ and regulating T-cell function. ²⁰

Based on the impressive wealth of data on brazilin, cytotoxic effects of Su Fu’ning Lotion (SFN), a lotion produced from sappan wood extract (with brazilin content >52%), on human bladder carcinoma cell line T24, mouse bladder transitional cancer cell line (BTT cells), and human bladder cancer cell line BIU-87 were assessed. Remarkable inhibitory effects of SFN on bladder tumor cell lines were found, and tumor cells died within 30 to 40 minutes of SFN treatment. In order to assess the in vivo inhibitory effects of SFN on bladder cancer, mouse models of bladder cancer were established using T24 and BTT xenografts. It was found that intravesical instillation of SFN significantly reduced mouse bladder wet weight and prolonged animal survival compared with controls (P < .05), suggesting that SFN significantly inhibited T24/BTT cell growth in mice.

These findings provide a basis for a new treatment method to reduce the recurrence rate of bladder cancer in patients after surgical treatment.

Materials and Methods

Cells and Culture Conditions

The human bladder cancer cell line T24 was purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Mouse bladder transitional cancer cell lines (BTT cells) and human bladder cancer cell line BIU-87 were a gift from Xiaofeng Yang, the director of Urology Surgery Department of the First Affiliated Hospital of Shanxi Medical University (Taiyuan, China).

The human bladder cancer cell line BIU-87 was first established from human bladder papillary transitional cell carcinoma in 1989. ²¹ The BIU-87 cells were usually inoculated subcutaneously to generate the bladder tumor animal model. ²² The doubling time of BUI-87 cell population (22nd generation) was 34 hours. ²³ BIU-87 cells could grow in soft agar media and its clone formation rate was about 23%. ²⁴

T24, BIU-87, and BTT cells were cultured in Roswell Park Memorial Institute Medium-1640 (Gibco, Life Technologies, Carlsbad, CA, USA) containing 0.05 g/L streptomycin, 0.05 g/L penicillin, 0.8 g/L sodium bicarbonate, 3.6 g/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Sangon Biotech Co, Ltd, Shanghai, China), and 10% fetal bovine serum (Sijiqing Co, Ltd, Hangzhou, China) at 37°C with 5% carbon dioxide and saturated humidity. Cells at logarithmic phase were selected for experiments.

Assessment of Cell Proliferation Using Methyl Thiazolyl Tetrazolium Method

A total of 4 × 10³ cells per well were seeded into 96-well plates in 100 µL culture media. After incubation at 37°C with 5% carbon dioxide for 24 h, media were discarded and fresh media containing 200 µl of SFN at final concentrations of 12.5, 25, 50, 100, 200, and 400 µg/mL were added to wells (experimental groups). Wells without SFN (control group) and without cells (zero group) were also included. Each group had 5 replicates. After 48 hours of incubation, 20 µL of 5 mg/mL methyl thiazolyl tetrazolium (MTT) was added into each well and incubated for an additional 4 hours. Then, the media were carefully aspirated, and 150 µL dimethyl sulfoxide was added to dissolve the blue-violet formazan crystals formed on deoxidation of MTT by succinate dehydrogenase in mitochondria of live cells. After shaking for 10 minutes at low speed, absorbance was read on a Sunrise microplate spectrophotometer (Tecan Inc, Grödig, Austria) at 570 nm. Growth inhibitory rate and inhibitory concentration (IC₅₀) were derived as follows:

$$\mathrm{Growth\; inhibitory\; rate}\left(\%\right)=\left(1-\mathrm{mean\; optical\; density}\left(\mathrm{OD}\right)\mathrm{value\; of\; the\; experimental\; group}/\mathrm{mean\; OD\; value\; of\; the\; control\; group}\right)\times 100\%.$$

$$\log {\mathrm{IC}}_{50}=\mathrm{Xm}-\mathrm{I}*\left(\mathrm{P}-\left(3-\mathrm{Pm}-\mathrm{Pn}\right)/4\right)$$

where Xm represents log (maximum dose); I represents the log (maximum dose/neighboring dose); P represents the sum of positive reaction rate; Pm represents the maximum of positive reaction rate; and Pn represents the minimum of positive reaction rate.

Similarly, chemotherapeutic drugs including mitomycin (MMC) and pirarubicin (THP) at different concentrations were served as positive controls, and respective growth inhibitory rates and IC₅₀s were determined.

Animals

The animal experimental protocol was approved by the local ethical committee and followed the guidelines of the National Research Council Guide for the Care and Use of Laboratory Animals.

The 5- to 6-week-old female inbred T739 mice (weighing 20-22 g) and 6- to 7-week old female BALB/c nude mice (weighing 20-22 g) were purchased from Beijing HFK Bio-Technology Co Ltd (Beijing, China) and Vital River Laboratories (Beijing, China), respectively. The animals were housed in a clean grade barrier environment of animal laboratory at 26° ± 1.5°C and 40% to 60% humidity. Animals were free access to sterilized drinking water and sterilized nutritional feed.

Establishment of Bladder Cancer Models

The mouse model used was described previously. ²⁵ Mice were anesthetized with sodium pentobarbital (60 mg/kg) by intraperitoneal injection, and hypogastrium was routinely disinfected. A 24-gauge venous indwelling needle cannula coated by liquid paraffin was slowly inserted into the bladder lumen through the urethral orifice and the urine was pulled out. Then, the core needle with deflection angle was slowly inserted into the cannula. The cannula was stabilized and the core needle was rotated for 5 rounds. Afterward, the core needle was pulled out and 100 µL of T24 or BTT cell suspension (1 × 10⁶ cells) was injected immediately. Finally, the cannula was pulled out; the animals woke up naturally. In the control group, no core needle was inserted to cause bladder mucosa injury after cannula insertion and the cell suspension was directly injected into the bladder lumen. The urinary bladders with injured and uninjured mucosa are represented in Figure 1.

Figure 1.

Comparison between injured mucosa and uninjured urinary bladder. (A) Uninjured mucosa; (B) injured mucosa. The bladders were injected with physiological saline.

Animal Treatments and Observations

Twenty-four hours after inoculation, mice were randomly divided into 4 groups, namely model, MMC, high-dose SFN, and low-dose SFN groups (n = 10 in each group). Mice were anesthetized with 60 mg/kg sodium pentobarbital (Sigma, St Louis, MO, USA) and the urethral orifice was locally disinfected. Infusion drugs were dissolved in 0.1 mL of water and injected into the bladder cavity. Then, cannulas were slowly pulled out and the urethral orifices were occluded using bulldog clamps for 30 minutes. The mice in model group were infused with normal saline. The mice in the MMC group were infused with 3.3 mg/kg MMC (MWanle Co, Ltd, Shenzhen, China) twice, on first and sixth days. The mice in the high- and low-dose SFN groups were infused with 120 and 60 mg/kg sappan wood extract (offered by Shanxi Cancer Hospital; Batch No. 20130518), respectively, once daily for 6 days.

Mice were housed conventionally after infusion, and observed at 08:00 and 20:00 hours every day. Animal survival was recorded over a period of 60 days: If death was observed at 08:00 hours, a full day survival was considered; a half day survival was considered if death was observed at 20:00 hours. Survival beyond 60 days was regarded as 60 days. The mice were dissected immediately after death and the bladder tumors were observed with the naked eye. Subsequently, the bladders were extracted and weighed. Since the bladder was lightweight and considering the difficulty involved in dissecting the tumor as such after the death of the mice, the whole bladder was weighed instead of tumor as such. Histological analyses were carried out on CX21FS1 (Olympus, Tokyo, Japan) and Leica DMIL 090-135.001 (Leica, Wetzlar, Germany) microscopes.

Statistical Analyses

Tumor formation rate was described as percentage (%). Survival time, tumor volume, and tumor weights were presented as mean ± standard deviation. All data were analyzed using the SPSS17.0 software (SPSS Inc, Chicago, IL, USA). After normality and homoscedasticity tests, comparisons between groups were performed using Kruskal-Wallis 1-way analysis of variance and the Bonferroni correction methods. Differences were considered statistically significant at P < .05.

Results

Higher SFN Concentrations Rapidly Exterminated Bladder Cancer Cells

Bladder cancer cells were treated in vitro with 3 drugs—SFN, MMC, and THP—at different concentrations, and the MTT method was used to assess cell viability. The 3 drugs displayed different toxicity on T24, BTT, and BIU-87 cells (Tables 1, 2, and 3, respectively). In T24 cells treated with 2 mg/mL SFN, cell proliferation inhibitory rates increased significantly with time, reaching 99.08% after 100 minutes; cell proliferation inhibitory rate of 100% was observed in T24 cells treated with 0.75 mg/mL THP in 20 minutes. In BTT cells, SFN and THP inhibited cell proliferation by 100% after 40 and 60 minutes, respectively. In BIU-87 cells treated with 2 mg/mL SFN, cell proliferation inhibitory rates increased significantly with time, reaching 93.46% after 100 minutes; cell proliferation inhibitory rate of 100% was observed in BIU-87 cells treated with 0.75 mg/mL THP in 80 minutes. At each time point, SFN and THP groups were significantly different (P < .05) compared with controls. However, no significant difference was observed between SFN and THP groups. Growth of the 3 cell lines was not significantly (P > .05) inhibited by 0.66 mg/mL MMC (Tables 1, 2, and 3).

Table 1.

Inhibition Rates in T24 Bladder Cancer Cells (%). ^a

Group	20 Minutes	40 Minutes	60 Minutes	80 Minutes	100 Minutes
CON	0.00 ± 0.00	0.41 ± 0.51	0.59 ± 0.32	0.85 ± 0.27	0.40 ± 0.52
SFN	5.89 ± 1.02	16.14 ± 0.81	75.81 ± 1.18*	91.46 ± 0.89*	99.08 ± 0.38*
MMC	0.59 ± 0.68	0.70 ± 0.54	1.35 ± 0.57	2.29 ± 1.06	3.01 ± 1.00
THP	100 ± 0.00*	100 ± 0.00*	100 ± 0.00*	100 ± 0.00*	100 ± 0.00*

Abbreviations: CON, control; SFN, Su Fu’ning; MMC, mitomycin; THP, pirarubicin.

a

Drugs used were SFN (2 mg/mL), MMC (0.66 mg/mL), and THP (0.75 mg/mL). Cell growth inhibition rate was defined as (1 − mean optical density [OD] value of the experimental group/mean OD value of the control group) × 100%. Data presented as mean ± standard deviation.

*

P < .05 compared with the control group.

Table 2.

Inhibition Rates in BTT Bladder Cancer Cells (%). ^a

Group	20 Minutes	40 Minutes	60 Minutes	80 Minutes	100 Minutes
CON	0.48 ± 0.68	0.55 ± 0.72	1.01 ± 0.81	0.92 ± 0.48	1.31 ± 1.00
SFN	90.35 ± 0.77*	100 ± 0.00*	100 ± 0.00*	100 ± 0.00*	100 ± 0.00*
MMC	2.81 ± 0.35	3.58 ± 1.11	4.07 ± 0.73	4.44 ± 0.94	4.55 ± 1.09
THP	8.91 ± 1.29*	97.72 ± 1.22*	100 ± 0.00*	100 ± 0.00*	100 ± 0.00*

Abbreviations: CON, control; SFN, Su Fu’ning; MMC, mitomycin; THP, pirarubicin.

a

Drugs used were SFN (2 mg/mL), MMC (0.66 mg/mL), and THP (0.75 mg/mL). Cell growth inhibition rate was defined as (1 − mean optical density [OD] value of the experimental group/mean OD value of the control group) × 100%. Data presented as mean ± standard deviation.

*

P < .05 compared with the control group.

Table 3.

Inhibition Rates in BIU-87 Bladder Cancer Cells (%). ^a

Group	20 Minutes	40 Minutes	60 Minutes	80 Minutes	100 Minutes
CON	0.82 ± 0.36	0.93 ± 0.40	1.11 ± 0.55	0.94 ± 0.37	0.91 ± 0.83
SFN	24.06 ± 2.20*	63.91 ± 4.49*	74.08 ± 13.28*	94.14 ± 1.10*	93.46 ± 1.03*
MMC	1.93 ± 0.23	2.80 ± 0.71	3.33 ± 0.17	4.37 ± 2.28	16.56 ± 6.43
THP	17.69 ± 9.04*	28.10 ± 5.22*	98.75 ± 0.87*	100 ± 0.00*	100 ± 0.00*

Abbreviations: CON, control; SFN, Su Fu’ning; MMC, mitomycin; THP, pirarubicin.

a

Drugs used were SFN (2 mg/mL), MMC (0.66 mg/mL), and THP (0.75 mg/mL). Cell growth inhibition rate was defined as (1 − mean optical density [OD] value of the experimental group/mean OD value of the control group) × 100%. Data presented as mean ± standard deviation.

*

P < .05 compared with the control group.

Determination of IC₅₀ Value of SFN on Bladder Cancer Cell Lines

To obtain the exact effective concentration, T24 cells were treated for 48 hours with an SFN concentration gradient. A cell proliferation inhibitory rate of more than 90% was observed with 25 μg/mL SFN, indicating that SFN was cytotoxic to T24 cells at low concentrations (Table 4). The IC₅₀ of SFN in T24 cells was determined to be 13.22 μg/mL, based on growth inhibitory data. The IC₅₀ value of 11.22 μg/mL was obtained for SFN in BTT cells (Table 5). Similarly, the IC₅₀ of SFN in BIU-87 cells was determined to be 12.5 μg/mL (Table 6).

Table 4.

Growth Inhibitory Rate of Different Concentrations of SFN in T24 cells (%). ^a

Group	OD Value	Inhibitory Rate (%)
CON	0.943 ± 0.036	—
SFN (µg/mL)
400	0.023 ± 0.007*	97.56
200	0.020 ± 0.001*	97.88
100	0.011 ± 0.002*	98.83
50	0.013 ± 0.007*	98.62
25	0.066 ± 0.015	93.00
12.5	0.518 ± 0.014	45.07

Abbreviations: CON, control; SFN, Su Fu’ning; OD, optical density.

a

Data presented as mean ± standard deviation. N = 5.

*

P < .05, compared with the CON group.

Table 5.

Growth Inhibitory Rate of Different Concentrations of SFN on BTT Cells (%). ^a

Group	OD Value	Inhibitory Rate (%)
CON	0.822 ± 0.027	—
SFN (µg/mL)
400	0.025 ± 0.004*	96.96
200	0.032 ± 0.005*	96.11
100	0.036 ± 0.002*	95.62
50	0.043 ± 0.007*	94.77
25	0.052 ± 0.006*	93.67
12.5	0.235 ± 0.023*	71.41

Abbreviations: CON, control; SFN, Su Fu’ning; OD, optical density.

a.

Data presented as mean ± standard deviation. N = 5.

*

P < .05, compared with the CON group.

Table 6.

Growth Inhibitory Rate of Different Concentrations of SFN in BIU-87 cells (%). ^a

Group	OD value	Inhibitory Rate (%)
CON	1.128 ± 0.019	—
SFN (µg/mL)
400	0.091 ± 0.013*	91.93
200	0.077 ± 0.007*	93.17
100	0.099 ± 0.016*	91.22
50	0.145 ± 0.021*	87.15
25	0.286 ± 0.030*	74.65
12.5	0.560 ± 0.066*	50.35

Abbreviations: CON, control; SFN, Su Fu’ning; OD, optical density.

a

Data presented as mean ± standard deviation. N = 5.

*

P < .05, compared with the CON group.

SFN Therapeutic Effects on Bladder Cancer

All BALB/c nude mice developed tumors after inoculation of T24 cells. Macroscopic tumors were found in bladder of some animals. Microscopic analyses showed that tumor cells had infiltrated into the muscular layers and proliferated outside the tissues as well. Thickened and hardened bladders, and small lumps of tumor cells, which extended to the mucous layers, were observed under a Leica 090-135.001 microscope. Some animals had tumor metastases in liver, kidney, mesentery, and ureter; in some mice, ascites and/or hematuria were noted. The average bladder wet weight in animals of the model group was 138.74 ± 145.57 mg, which was significantly higher than in the experimental group (P < .05). No significant difference was found among the MMC, and high- and low-dose SFN groups. The average animal survival time in the model group was 30.20 ± 8.35 days, which was significantly lower (P < .05) than that observed in the high-dose SFN group (43.50 ± 8.11 days). No significant difference was observed between the MMC group and model group. The rate of tumor transformation was 20% in the model group, while it was 10% in the low-dose SFN group. Importantly, no tumor transformation was observed in the high-dose SFN and MMC groups (Table 7).

Table 7.

Average Lifetime, Bladder Wet Weight, and Tumor Metastasis in BALB/c Nude Mice Inoculated With T24 Cells.

Group	n, Beginning/End	Dosage (mg/kg) × Times	Average Lifetime (Days)	Average Bladder Wet Weight (mg)	Tumor Metastasis (%)
Model	10/0	—	30.20 ± 8.35	138.74 ± 145.57	20
MMC	10/0	3.3 × 2	34.60 ± 10.36	39.92 ± 20.42*	0
High-dose SFN	10/1	120 × 6	43.50 ± 8.11*	39.78 ± 29.34*	0
Low-dose SFN	10/1	60 × 6	39.80 ± 12.97*	38.61 ± 29.10*	10

Abbreviations: SFN, Su Fu’ning; MMC, mitomycin.

a

Animals in the model group were infused with normal saline. In the MMC group, mice were infused with 3.3 mg/kg MMC twice, on first and sixth days. High- and low-dose SFN group animals were infused with 120 and 60 mg/kg SFN, respectively, each day, for 6 days. Data presented as mean ± standard deviation.

*

P < .05, compared with the model group.

All T739 mice developed macroscopic large tumors after inoculation of BTT cells (100% tumor formation rate). There was no significant difference in bladder wet weight between each group. Some animals had tumor metastases in liver, kidney, mesentery, and ureter; in some mice, ascites or hematuria was noted. The average survival time was 25.7 ± 4.5 days in the model group, which was significantly lower (P < .05) than that observed in the high-dose SFN group (40.0 ± 14.0 days). However, no significant difference was found while comparing the other experimental groups and the model group (Table 8). The rates of tumor transformation were 50%, 20%, 30%, and 20% in the model, high-dose SFN, low-dose SFN, and MMC groups, respectively.

Table 8.

Average Lifetime, Bladder Wet Weight, and Tumor Metastasis in T739 Mice Inoculated With BTT Cells. ^a

Group	n, Beginning/End	Dosage (mg/kg) × Times	Average Lifetime (Days)	Average Bladder Wet Weight (mg)	Tumor Metastasis (%)
Model	10/0	—	25.70 ± 4.50	459.95 ± 173.67	50
MMC	10/0	3.3 × 2	24.80 ± 6.66	262.67 ± 139.43*	20
High-dose SFN	10/1	120 × 6	40.00 ± 14.09*	528.43 ± 163.57	20
Low-dose SFN	10/1	60 × 6	33.40 ± 12.18	536.39 ± 200.96	30

Abbreviations: SFN, Su Fu’ning; MMC, mitomycin.

a

Animals in model group were infused with normal saline. In the MMC group, mice were infused with 3.3 mg/kg MMC twice, on first and sixth days. High- and low-dose SFN group animals were infused with 120 and 60 mg/kg SFN, respectively, each day, for 6 days. Data presented as mean ± standard deviation.

*

P < .05, compared with the model group.

These results indicated that SFN significantly inhibited the growth of tumor cells, reduced tumor metastasis, and prolonged the survival time in mouse tumor transplantation model of bladder cancer.

Discussion

Bladder cancer is a huge burden to public health considering its incidence and recurrence rates ² and intravesical instillation is considered one of the most important measures used to prevent postoperative recurrence and reduce mortality of patients. ³

In the present study, in vitro data showed that SFN was highly effective at low concentrations against mouse (BTT) and human bladder cancer cell lines (T24 and BIU-87), with IC50 of 13.22, 11.22, and 12.5 μg/mL for T24, BTT, and BIU-87 cells, respectively. Based on these data, it was hypothesized that SFN could be active in vivo in the treatment of bladder cancer. Hence, a mouse bladder tumor model was developed by orthotopic transplantation of T24/BTT xenografts using the angle needle method. The mouse bladder tumor model was established using a simple surgical approach with advantages of high tumor formation rates and little injury to animals. The results showed that intravesical instillation of SFN (containing brazilin >52%) effectively inhibited the bladder tumor growth and prolonged the survival time of animals.

Although bladder wet weight of the MMC group was reduced compared with the model group, the average survival time of the animals did not increase in this experimental group, which probably because of the toxic secondary effects of MMC. The average bladder wet weight was similar in T739 mice for all groups. However, the average survival time was significantly increased in the high-SFN group and the tumor formation rate was lower in comparison with the model group, indicating that high SFN concentrations possess inhibitory effects on tumor cell growth. Overall, SFN was superior to MMC (positive control) in terms of therapeutic effects and safety after intravesical instillation.

Brazilin (in SFN) has been reported to have many biological activities, including immune regulation,^19,20 blood vessel dilatation,^3,11,14 anticoagulation,^8,17 hypoglycemic effect,^{10,13,15,16,26,27} oxidative damage inhibition,^6,28 antibiosis, ⁹ and anti-inflammatory effects.^4,5,7 In addition, it was demonstrated that brazilin powerfully broke deoxynucleic acid chains in anticancer screening. ¹² It was previously reported that broken nucleus and dispersed chromatin were found in human ovarian cancer cells (AKOV3) after treatment with sappan wood extract, indicating cancer cell apoptosis.^{29 -31} Further studies to explore the mechanism of SFN are required.

Another key feature of brazilin is its low toxicity. Indeed, Hu et al ⁹ demonstrated that brazilin had no toxicity effects on normal cells at concentrations lower than 300 μmol/L. Interestingly, in the present study, an IC₅₀ of 13.22 μg/mL (46.22 μmol/L) was established for brazilin in human bladder cancer cells T24. In addition, animals dosed with 120 mg/kg brazilin showed no apparent toxicity. Thus, brazilin should be considered a safe and effective antitumor constituent, whose potential value in the prevention and treatment of bladder cancer deserves further exploration.

Conclusion

Su Fu’ning Lotion inhibits bladder cancer cell proliferation in vitro and in vivo and significantly prolong the survival of mice with bladder cancer xenografts, indicating that SFN could be used as a perfusate after surgery for bladder cancer removal to reduce tumor recurrence.