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Capsaicin-Induced Apoptosis in MBT-2 Murine Bladder Tumor Cells and Bladder Wall Penetration Effect of the Nano-Encapsulated Capsaicin in Rabbit

Purpose: In this study, we attempted to characterize capsaicin’s effects with regard to the apoptosis of murine bladder cancer cells (MBT-2) as well as the pharmacodynamics of nano-encapsulated capsaicin formulation for intravesical instillation. Materials and Methods: We assessed the viability of the MBT-2 cells via MTT staining, agarose gel electrophoresis, and flow cytometric apoptosis analysis. Intravesical reagents were instilled into 3 groups of male white New Zealand rabbits. Instillation agents were nano-encapsulated capsaicin dissolved in saline, capsaicin dissolved in saline, and capsaicin dissolved in dimethyl sulfoxide (DMSO). We also determined the pharmacokinetics of urine, plasma, and bladder tissue after intravesical capsaicin instillation. Results: Capsaicin treatment was determined to reduce cell viability in a timeand dose-dependent manner. The capsaicin concentrations in the urine of the rabbits decreased in each of the treatment groups, but we noted a more profound reduction of capsaicin concentration in the nano-encapsulated capsaicin group. Plasma concentrations were definitely lower as compared with the levels measured in the bladder tissue and urine. We noted distinctive differences in patterns of concentration change between the capsaicin with normal saline solution (NSS) or DMSO and the nano-encapsulated capsaicin groups. The concentration of nano-encapsulated capsaicin in the tissue appeared to increase directly with tissue depth. Conclusions: Our results show that capsaicin can induce apoptosis in MBT-2 cells, as well as the excellent permeation properties of nano-encapsulated capsaicin. Treatment with intravesical capsaicin may be a promising alternative therapeutic modality for the treatment of bladder cancer. (Korean J Urol Oncol 2019;17:168-177) 󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏󰠏


INTRODUCTION
Bladder cancer is the most frequently encountered urological malignancy in Korea. Approximately 70% of newly diagnosed bladder cancers are superficial variants (Ta, T1, or Tis). 1 The normal treatment regimen for these superficial bladder cancers includes a transurethral resection for the removal of any visible tumors, coupled with subsequent intravesical therapy. This technique has been widely considered to be the most effective method for the prevention of bladder cancer recurrence, as well as the aggravation to invasive bladder cancer. Intravesical chemotherapy has the decided advantage of being able to selectively deliver high concentrations of drugs to the tumor-bearing bladder, while minimizing overall systemic exposure. Intravesical bacillus Calmette-Guerin (BCG) constitutes the main treatment modality currently used in cases of superficial bladder cancer.
However, 30%-40% of treated patients fail to respond to BCG, and another 30%-40% of initial responders experience a relapse within 5 years. BCG has also been associated with possibly severe complications, including fever, granulomatous prostatitis, hematuria pneumonia, sepsis, and hypersensitivity.
In previous studies, a host of intravesical chemotherapeutic agents have been assessed, and many have been shown to reduce short-term recurrence rates. However, none have proven effective for preventing long-term recurrence rates or tumor progression. Several studies have reported BCG's superior efficacy as compared to mitomycin C and doxorubicin. [2][3][4][5] Several studies have also indicated that the failure mitomycin C and doxorubicin treatments are attributable, in part, to the inability of the drugs to penetrate bladder tissue and also to the low activity of these drugs when pitted against more aggressive tumors. [6][7][8][9][10] Enhanced drug delivery was shown to improve treatment outcomes in a phase III trial, which demonstrated that the augmentation of mitomycin C delivery resulted in a recurrence-free rate nearly double previous levels in superficial bladder cancer patients. 11 Taken together, these findings show that the 2 most important properties of a drug to be used in intravesical therapy are its activity against bladder cancer and its ability to penetrate bladder tissues. As discussed below, capsaicin appears to be an attractive candidate for such use.
Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the primary acrid compound in the red pepper, has been applied in food ingredients and drugs for quite a long time.
Recently, an attempt was made to use capsaicin as an intravesical drug for the treatment of detrusor hyperreflexia in overactive bladder. 12 Capsaicin has also been demonstrated to impair growth and cause apoptosis in vitro in a variety of transformed cell types, but this was not found in normal counterpart cells. [13][14][15][16] Furthermore, these compounds re-portedly promote apoptosis in vivo as the mechanism underlying tumor cell elimination in animal carcinogenesis models. 17 In particular, studies have shown that in certain bladder cells capsaicin induces cell cycle arrest, apoptosis, and decreased cell migration. 18 Chemically, capsaicin features an aromatic ring, an amide bond, and a hydrophobic side chain in its structure, which renders it an extremely hydrophobic molecule that is insoluble in water. Therefore, for intravesical application, capsaicin is normally incorporated with normal saline solution (NSS) and 30% ethanol. 19 However, this vehicle has been reported to exacerbate the adverse histological changes associated with capsaicin, including epithelial thinning and submucosal edema. 20 Thus, better and safer vehicles for water-insoluble capsaicin are clearly a prerequisite for the development of an effective delivery protocol.
In order to improve the delivery of the extremely hydrophobic capsaicin, we decided to encapsulate it within a hydrophobic polymer matrix. Therefore, we selected polylactic acid-polyglycolic acid (PLGA) copolymer and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (Pluronic F-127) as materials for the encapsulation. 21 In the present study, in order to assess the possibility of

3) DNA fragmentation detection
Following incubation, the cell that were either treated with or without capsaicin, were harvested via centrifugation

4) Flow cytometric analysis
In order to confirm the extent and rate of apoptosis, the cells were then stained with propidium iodide as previously described. 23 The cells were then examined applying flow cytometric analysis using the FACSCalibur system (Becton-Dickinson, Franklin Lakes, NJ, USA) equipped with the Cell Quest program.

5) Statistical analysis
Two-tailed Student t-tests were used in order to examine the significance of the tested data. The data were demonstrated as the means±SEM. Values of p＜0.05 were considered to be significant.

1) Materials
Pluronic F-127 can be represented by the formula poly Tween 80, and capsaicin were purchased from Sigma.

Nanoshperes
Ten ml PLGA/tetraglycol solution was prepared separately by mixing PLGA with tetraglycol. Then, 0.5% (by weight) Tween 80 was added to the total amount of PLGA as an emulsifier. This solution was then sprayed into 100-mL water, in order to induce the precipitation of PLGA

3) Animal study
This study was performed with 15 male white New Zealand rabbits, each weighing between 2-3 kg (Changddul, Gwangju, Korea). These rabbits were given access to both water and feed ad libitum. The rabbits were exposed to light from 7 AM to 6 PM, and their environment was maintained at a temperature of 22℃-24℃ and a humidity of 40%-60%. The collected blood samples were also frozen immediately with liquid nitrogen. The plasma capsaicin concentrations, however, were so extremely low that we used competitive inhibition enzyme immunoassay, with a measurement limit of 0.2 ng/mL.

(3) Capsaicin concentrations in bladder tissue
Frozen bladder wall tissue samples were carved, parallel to the urothelial surface, into 50-m slices using a cryotome After 15 minutes of centrifugation at 2,000×g at 4℃, the aqueous supernatant was extracted by suction and removed.
The remaining organic layer was then quantitatively moved to a 10-mL glass tube. The residue compound was recon-   and 100 L of the reconstructed derivatized product was analyzed via HPLC. The first sections were abandoned, in order to dismiss any tissue contamination due to urine. The next 8 sections were then individually analyzed, whereas the latter sections were pooled for analysis as they contained lower drug concentrations.

5) Statistical analysis
We employed 1-way analysis of variance tests and post hoc multiple comparisons in order to evaluate the significance of the data. The data were expressed as means± standard error of the mean. Values of p＜0.05 were considered to be significant.

The Effects of Capsaicin in MBT-2 Cells
Capsaicin's effects on the viability of MBT-2 cells were determined via MTT staining. Capsaicin was found to reduce cell viability in a dose-dependent manner (Fig. 1).
Capsaicin began to evidence significant cytotoxicity at a concentration of 100 mM. In order to determine the extent to which capsaicin induced apoptotic cell death in MBTmurine bladder cancer cells, we conducted 2 independent measurements, using agarose gel electrophoresis and flow cytometry. We determined that capsaicin induced a dose-dependent DNA fragmentation-a hallmark of apoptosis-as demonstrated by the results of agarose gel electrophoresis (Fig. 2). Furthermore, capsaicin (100 M) was also shown to induce apoptosis in a time-dependent manner, according to the results of flow cytometry, in which the hypodiploid DNA content was determined via propidium iodine staining (Fig. 3). These results demonstrate that capsaicin exerts an apoptosis-inducing effect in MBT-2 human bladder cancer cells.  ward trend in urine concentration from zero time to 10 minutes reflects the dilution of capsaicin by residual urine in the bladder. As the therapy progressed, continuing urine accumulation also effected an increased dilution, and hence a further decrease in the concentration of the drug. We also noted a significant difference between the capsaicin dissolved in NSS or DMSO and the nano-encapsulated capsaicin after 30 minutes of treatment (p＜0.001). The nanoencapsulated capsaicin concentration declined at a more rapid rate than did the concentrations of the NSS or DMSOdissolved capsaicin.

2) Capsaicin concentration changes within the blood
Capsaicin concentrations within the plasma were determined to increase over time, but these concentrations remained extremely low overall (Fig. 4B). We found no significant differences among the experimental groups in this regard (p＞0.3).

DISCUSSION
In multicellular organisms, differentiated cells apparently share the ability to induce their own death via the activation of an internally encoded suicide program, which is commonly referred to as apoptosis. Apoptosis is arguably the most potent natural defense against the development of cancer. Therefore, many attempts have been made to develop a chemoprevention strategy that selectively triggers apoptosis in both malignant and premalignant cancer cells.
In this study, we have evaluated the possibility of using  Before the present study, there had been no definitive histopathological analyses of the changes in bladder mucosa as the result of intravesical capsaicin application. In a previous study, it was demonstrated that acute damage to urothelial integrity was more profound in rats treated with capsaicin in ethanol than in the rats which had been treated solely with an ethanol vehicle. However, the urothelial tissue in these rats returned to normal within a few days. 39 Hence, we conclude that the intravesical instillation of nano-encapsulated capsaicin may constitute a promising alternative therapeutic modality, and the further evaluation of the properties and efficacy of nano-encapsulated capsaicin in superficial bladder tumor models is clearly warranted.