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泌外梗阻研究生郭鹏荣The investigation of a traditional Chinese medicine, Guizhi Fuling Wan (GFW) as an intravesical therapeutic agent for urothelial carcinoma ofthe bladder•Abstract•Background:The high risk of recurrence faced by patients with bladder cancer has necessitated the administration •of supplemental intravesical chemotherapy; however, such treatments often result in severe side effects. As a result,novel intravesical agents with enhanced efficacy and minimal toxicity are urgently required for the treatment ofbladder cancer.•Methods:Guizhi Fuling Wan (GFW) is a traditional Chinese medicine shown to inhibit the growth of hepatocellular •carcinoma. This study evaluated the growth inhibition of GFW using normal human urothelial cells and bladder •cancer cells; the efficacy of GFW treatment was further compared with mitomycin C, epirubicin, and cisplatin. We •also examined the progression of cell cycle and apoptosis in bladder cancer cells in response to GFW treatment.•CCK-8 was employed to analyze cell viability and flow cytometry was used to study the cell cycle and apoptosis.•The mechanisms underlying GFW-induced cell cycle arrest were determined by Western blot analysis.•Results:Our data demonstrate the potent inhibitory effect of GFW in the proliferation of bladder cancer cell lines,•BFTC 905 and TSGH 8301. GFW presented relatively high selectivity with regard to cancer cells and minimal toxicityto normal urothelial cells. Our results also demonstrate that GFW interferes with cell cycle progression through the activation of CHK2 and P21 and induces apoptosis in these bladder cancer cells.•Conclusions:Our results provide experimental evidence to support GFW as a strong candidate for intravesicle •chemotherapy against bladder cancer•Background•Urothelial carcinoma (UC) is the most common cancer of the urinary tract [1] and the fourth most common•malignancy in the U.S. [2]. The most common histological type of UC is transitional ce ll carcinoma (TCC),•which is originated from the urothelial lining of the urinary tract [3]. Although UC may occur anywhere in•the urinary tract, it generally originates in the urinary bladder [4]. Bladder cancer patie nts diagnosed with nonmuscle invasive disease have a high risk of recurrence [5], ne cessitating the use of intravesical chemotherapy or Bacillus Calmette-Guerin (BCG) a s a supplement totransurethral resection (TUR) [6]. Unfortunately, intravesicalchemot herapy, such as mitomycin C or thiotepa, commonly produce severe side effects, incl uding urinary frequency, urinary urgency, cystitis, and hematuria [7].Thus, novel intrav esical agents with proven efficacy and minimal toxicity are urgently required for the tr eatment of bladder cancer.Guizhi Fuling Wan (GFW) is a well-known traditional Chine se herbal formula, comprising five herbs includingCinnamomi Ramulus, Poria Cocos, Paeoniae Radix Rubra,•Persicae Semen, and Moutan Cortex [8].•Due to its sedative, analgesic, and antiinflammatory effects, GFW has also been used in thetreatment of various diseases. For example, GFW hasbeen shown toinhibit the growth of hepatocellular carcinoma [12,13] and cervical cancer [14].However, theeffect of GFW on urothelial carcinoma has never beenexplored. This study compared the effects of GFW withvarious other chemotherapeutic agents in the growth ofnormal human urothelial cell and two cancer cell lines.The effects of these agents on cell cycle progression andapoptosis in urothelial cancer cells were also compared.Finally, we sought to reveal the underlying mechanismsinvolved in cell cycle arrest induced by GFW.MethodsPreparation of agents and cell culturesGFW herbal extract (batch No. 221141) was purchasedfrom Sun Ten Pharmaceutical Co., Ltd. (Taichung City,Taiwan) and validated using HPLC as outlined in theSupplemental Experimental Procedures (see Additionalfile 1). GFW and Ramulus Cinnamomi(otherwise knownas Guizhi) (Sun Ten Pharmaceutical, Taiwan, ROC) weredissolved in ddH2O and filtered using a 0.22 micronfilter at 220 mg/ml and 129.4 mg/ml,respectively. Theconcentrations of these stock solutions were thenconfirmed byweighing after lyophilization. Mitomycin-C(Kyowa Hakko Kirin Co., Tokyo, Japan), Epirubicin (ActavisItaly S.P.A., Milano, Italy) and Cisplatin (ABICBiologicalLaboratories Ltd., Netanya, Israel) were dissolved in normalsaline buffer at1 mg/ml to provide stock solutionswhich were then diluted with cell culture mediumtodesired concentrations ranging from 0.0025 to 0.08 mg/ml.Human bladder papillary transitional cells, BFTC 905,and bladder carcinoma cells, TSGH 8301 (FoodIndustryResearch and Development Institute, Taiwan, ROC) aswell as primary normal urothelial cells, HUC 4449(ScienCell Research Laboratories, Carlsbad, CA,•USA)were used as cell models. BFTC 905 and TSGH 8301were cultured in RPMI 1640 supplemented with 10%fetal bovine serum (FBS), 100 units/ml penicillin and100 μg/ml streptomycin. HUC 4449 was cultured inurothelial cell medium (ScienCellResearch Laboratory,Carlsbad, CA, USA) supplemented with 10% FBS, 100units/ml penicillin and 100μg/ml streptomycin. All celllines were cultured at 37°C under a humidified atmosphere containing 5% CO2. All studies involving humancell lineswerconformed to the HelsinkiclarationandapprovedbytheInstitutionalReviewBoardsoftheChia-Yi Christian Hospital (reference number: 099078).Cell viability assayBFTC 905, TSGH 8301 and HUC4449 were initiallyseeded in 96-well plates at 1 ×104cells per well andcultured for 24h. The cells were subsequently starvedin medium supplemented without FBS for 24 h,and thentreated with the agents of interest at various concentrationsfor 24 h. Cell viability was then determined using theCell Counting Kit-8 (CCK-8) (SIGMA,Switzerland), inaccordance with the manufacturer’s protocol.•In brief,the assay was performed using WST-8, [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2Htetrazolium, monosodium salt], which can be bio-reducedby cellular dehydrogenases to an orange formazanproduct, which is then dissolved in cell culture medium.The production of formazan occurs only in living cellsat a rate proportional to the number of living cells. Afterincubating the cells with CCK-8, the light absorbanceof the culture medium in each well was measuredat450/655 nm using Microplate reader Model 680 (BIORAD). The cell viability rate, compared to the control (untreated cells), was calculated using the following equation.•ResultsHPLC analysis of Guizhi Fuling Wan (GFW)Guizhi Fuling Wan is composed of five Chinese herbs,including Poria cocos, for which noaccepted watersoluble bioactive component is currently available.Thebioactive components of Cinnamomi Ramulus, PaeoniaeRadix Rubra, Persicae Semen, and Moutan Cortex arecinnamaldehyde, paeoniflorin,amygdalin, and paeonol,respectively. The structural variety of these four compoundsproduces various absorption wavelengths (see Additionalfile 1: Figure S1). A photodiode array detector providedsimultaneous detection of the compounds as follows: amygdalin (215 nm), paeoniflorin (232 nm),cinnamaldehyde(280 nm), and paeonol (275 nm) (Figure 1A). Thecalibration equations and correlation coefficients revealedlinearrelationships between the peak areas and concentrations (see Additional file 1: Table S1). The relative standard deviation (RSD) of these fourcompounds in GFWfell between 1.54-3.27% (intra-day) and 1.17-2.34%,(inter-day) (Table 1), indicating the high degree of reproducibilityassociated with this method. The contents ofthe compounds in GFW were as follows: amygdalin(11.53 mg/g),•paeoniflorin (232 nm), cinnamaldehyde(280 nm), and paeonol (275 nm) (Figure 1A).The calibration equations and correlation coefficients revealedlinear relationships between the peak areas and concentrations (see Additional file 1: Table S1). The relative standard deviation (RSD) of these four compounds in GFWfell between 1.54-3.27% (intra-day) and 1.17-2.34%,(inter-day) (Table 1), indicating the high degree ofreproducibility associated with this method. The contents ofthe compounds in GFW were as follows: amygdalin(11.53 mg/g), paeoniflorin (30.26 mg/g),cinnamaldehyde(0.10 mg/g), and paeonol (0.28 mg/g) (Table 1, Figure 1B).Maximum tolerated dosage of GFW in normal urothelialcellsA fundamental requirement ofintravesical agents is minimal cytotoxicity toward normal cells. To determinethemaximum dosage of GFW that normal urothelial cellscould tolerate, normal human urothelial HUC 4449 cellswere treated with various concentrations of GFW anditsmajor component, Ramulus Cinnamomi (Guizhi). Thenon-toxic effects of Guizhi have been demonstrated inMRC-5 normal lung epithelial cells [16]; therefore,weselected Guizhi as a negative control for this study.According to CCK-8 assay, GFW and Guizhi causedsignificant death (p<0.01) of HUC 4449 cells atconcentrations of 4 mg/ml and 8 mg/ml, respectively,compared to the untreatedcontrol (Figure 2). Thissuggests that GFW and Guizhi have cytotoxic effectsonnormal urothelium only at high concentrations.•Wetherefore calculated that a concentration of 2 mg/mlwould be the maximum safe dose that normal urothelialcells could withstand. This value was applied to all subsequent experiments dealing with cell cycle and apoptosis.Cytotoxic effects of GFW, cisplatin, epirubicin, andmitomycin-C on normal urothelial cells and bladdercancer cell linesNext, we performed anin vitro cell viability assay tocompare the cytotoxicity of GFW with traditionalchemotherapeutic agents(mitomycin C, epirubicin, andcisplatin) in normal urothelial andcancer cell pared with the untreated control, the CCK-8 assayrevealed that the cytotoxicity of GFW to TSGH 8301and BFTC 905 urothelial cancer cells is comparable tothat of the three other chemotherapeutic agents withdifferent IC50values (P< 0.001, one-way ANOVA) (Table 2and Figure 3). Traditional chemotherapeutic agents havemuch lower IC50towards TSGH 8301 and BFTC 905 cancer cells; however, even such low doses are toxic tonormalurothelial cells (Table 2 and Figure 3).中国传统的医学调查桂枝五涪陵Wan(GFW)作为一个移行细胞膀胱癌膀胱内的治疗药剂•背景:面临的高复发风险的患者膀胱癌症需要政府补充膀胱内的化疗;然而,这种治疗方法常常导致严重的副作用。