Background
Bladder cancer is one of the common cancer in the United States and many other parts of the world [
1,
2]. The estimated numbers of new cases are expected to reach 74,000/year in 2015 in the United States [
2]. Previous studies showed that approximately 30% of newly diagnosed superficial bladder cancers were multifocal, however, 60–70% of these superficial bladder cancers recurred, and 10–20% of them would undergo stage progression to a muscle-invasive or metastatic disease [
3]. The effect of chemotherapy or other systemic treatment for bladder cancer is limited [
4]. Although Platinum-based chemotherapy is commonly used for bladder cancer [
5,
6], radical cystectomy and systemic chemotherapy are suggested for invasive bladder cancer. These treatment regimens usually fail in 95% patients: at least half of the invasive bladder cancer patients still die of metastases within 2 years after diagnosis, and with less than 10% 5-year survival rate [
2,
7].
Several nutrients and non-nutritive phytochemicals have been evaluated in interventional trials for their potential as cancer chemo-preventive agents. Non-traditional treatments using herbs and dietary supplements have also been considered as alternative therapy for cancer.
Angelica sinensis (Oliv.) Diels (Umbelliferae), which is pronounced as “Danggui” in Mandarin, is one of the most commonly used herbs in traditional Chinese medicine (TCM). It is clinically administrated to replenish blood and to treat several gynecological symptoms such as menstrual disorders in women.
N-butylidenephthalide (BP), an active compound isolated from the chloroform extract of
Angelica sinensis, has been identified as having growth-inhibitory and apoptosis-induction effects on various cancer cells [
8‐
13]
. These findings highlight the potential therapeutic role of BP in clinical application. However, the effect of BP on human bladder cancer cells is still unclear and worth further investigation.
Our aim in this study was to investigate the probable anti-proliferative effect of BP on bladder cancer cells, and to determine the signaling pathway that might involve. In addition, NOD-SCID mice xenograft tumor model was used to evaluate the antitumor effect of BP on bladder cancer in vivo. On the other hand, since Taiwan’s National Health Insurance Research Database (NHIRD) has been successfully used in epidemiological studies of cancer and Chinese herbal products (CHPs) [
14,
15], we also investigated the correlation of taking
Angelica sinensis and the incidence of bladder cancer in Taiwan.
Methods
Cell proliferation assay, western blot and cell cycle analysis were performed as previously described [
9], with further details were provided in Additional file
1. For RNA isolation, cell migration and invasion assay, and quantitative RT-PCR, see Additional file
1.
Cell culture
Human bladder cancer cell line TCCSUP was purchased from ATCC (American Type Culture Collection, Manassas, VA). Human bladder cancer cell lines 5637, T24, and BFTC (BFTC 905) were purchased from BCRC (Bioresource Collection and Research Center, Hsinchu, Taiwan). Cells were cultured in appropriate culture medium and supplements according to the suggestion of ATCC and BCRC, respectively. Cell lines were authenticated annually by short-tandem repeat analysis and routinely tested for mycoplasma contamination (BCRC).
Chemicals and antibodies
BP (C12H12O2, 95%) was purchased from Lancaster Synthesis Ltd. (Newgate Morecambe, UK). Cisplatin, dimethyl sulfoxide (DMSO), [3-(4,5-dimethyl thizol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), crystal violet, DSD, Tween-20, methanol, and horseradish peroxidase-conjugated secondary antibodies were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The primary antibodies were all purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). Polyvinyldifluoride (PVDF) membranes, BSA protein assay kit and chemiluminescence reagents were purchased from Amersham Biosciences (Arlington Heights, IL, USA).
TUNEL assay
Human bladder cancer cells were cultured in the presence or absence of BP (60 μg/ml) for 72 h and then examined for apoptosis with TUNEL assay (In Situ Cell Death Detection Kit, Roche) according to the manufacturer’s instructions.
Annexin V-FITC staining
Human bladder cancer cells were cultured in the presence or absence of BP (60 μg/ml) for 3, 18 and 24 h, as indicated. The vehicle control group was treated with 0.2% DMSO only. Apoptotic cell death was examined using annexin V-FITC detection kits according to the manufacturer’s instructions (BD Biosciences, San Diego, CA, USA). Ten thousand events were acquired for each sample and analyzed by Accuri C6 flow cytometer with CFlow® software.
Animal studies
Tumors were generated as previously described [
9] and further details were provided in Additional file
1.
Patients and study design
Taiwan implemented a National Health Insurance (NHI) program in 1995 to provide comprehensive health care coverage. Enrollment in this government-run, universal, single-payer insurance system is mandatory and up to 99% of the 23 million residents of Taiwan receive medical care through the NHI program. In addition, >97% of the hospitals and clinics in Taiwan are contracted to provide health care services under the NHI [
16]. All data related to these services are collected and input into the NHIRD by the National Health Research Institute to provide a comprehensive record of medical care. The data consist of ambulatory care records, in-patient care records, and registration files of the insured, and the database includes all claims data from the NHI program. The NHI Bureau randomly reviews the charts of one out of every 100 ambulatory cases and one out of every 20 in-patient cases, as well as conduct patient interviews to verify the accuracy of the diagnosis [
17].
This study used the NHIRD collected within 2003 to 2009. The study design featured a study cohort and a comparison cohort. Patients with newly-diagnosed with bladder cancer (International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) code 188.XX) before the index date were excluded. Patients who received CHPs containing Angelica sinensis between 2002 and 2009 were then identified as the study cohort (the exposure group). The date of initiation of Angelica sinensis exposure was used as the patient’s index date. The control cohort (the non-exposure group) included all of the other patients without taking CHPs therapy, and propensity score matching with age and gender was applied to select the controls.
Charlson Comorbidity Index Score
The Charlson Comorbidity Index Score (CCIS) is a widely accepted measure for risk adjustment in administrative claims data sets [
18‐
20]. The CCIS were calculated for each patient by assigning 1 point each for myocardial infarct, congestive heart failure, peripheral vascular disease, dementia, cerebrovascular disease, chronic lung disease, connective tissue disease, ulcer, chronic liver disease and diabetes by assigning 2 points each for hemiplegia, moderate or severe kidney disease, diabetes with end organ damage, tumor, leukemia and lymphoma, 3 points for moderate or severe liver disease and 6 points each for malignant tumor, metastasis and acquired immune deficiency syndrome.
Other variables
The study subjects were classified into three groups: (1) low SES, < 583 US $ per month (New Taiwan Dollars [NTD] ~17,500); (2) moderate SES, 583–833 US $ per month (NTD 17500–25,000); and (3) high SES, ≥ 833 US $ per month (≥ NTD 25001) [
21]. Low income was set at NTD 17500 because this was the government-stipulated minimum wage for full-time employees in Taiwan in 2009. The geographic regions where the individuals resided were recorded as northern, central, southern, and eastern Taiwan.
Statistical analysis
All data were shown as mean ± S.D. Statistical differences were analyzed using the Student’s t-test for normally distributed values and by nonparametric Mann–Whitney U-test for values with a non-normal distribution. Significant differences between groups were evaluated using analysis of variance (ANOVA) with Games-Howell test as post-hoc test. Pearson’s chi-square test was used for categorical variables such as gender, SES, geographic region of residence, co-morbidities and incidence of bladder cancer.
Discussion
NHIRD is a population-based dataset which provides researchers to trace the medical service history covering over 99% population in Taiwan, and is widely recognized to provide highly accurate diagnoses and clinical information [
22,
23]. Several reports have used the NHIRD to evaluate the associations between different diseases [
14,
19,
20]. As there were considerable differences in age, gender and comorbidities between the patients who has or has not been exposed to
Angelica sinensis, propensity score matching with age and gender was applied to select the controls. The incidence of bladder cancer decreased in the patients who were exposure to
Angelica sinensis using a matching method. However, the limitations of this study data from NHIRD should, however, be noted. One obvious limitation study is that, the incidence of bladder cancer was low and the numbers of bladder cancer were likely too small for these types of sub-analyses. The other limitations from NHIRD were described in previous studies [
19,
20]. Briefly, the severity of bladder cancer cannot be precisely extracted from ICD-9-CM codes and the database does not contain information on tobacco use, dietary habits, carcinogen exposure and body mass index, which may also be risk factors for bladder cancer were the limitations. In addition, the CHPs used might contain additional herbs other than
Angelica Sinensis and additional bio-active agents. In order to clarify the results from the NHIRD study and the physiological effect of BP on bladder cancer cells, we thus carried out the in vitro and in vivo experiments.
Many dietary phytochemicals and extracts derived from herbs have been tested as antioxidants, or as inhibitors of cancer cell proliferation in vitro and in vivo [
24]. Recent reports have demonstrated that BP is cytotoxic to various cancer cells including glioblastoma multiforme (GBM), hepatocellular carcinoma (HCC) and prostate cancer. However, the effect of BP on human bladder cancer cells has not been addressed. Based on the abovementioned discovery that patients who have been exposed to
Angelica sinensis have lower incidence of bladder cancer, we thus intended to study the probable physiological effect of BP on bladder cancer using bladder cancer cell lines of various stages.
In the subsequent study, we demonstrated the cytotoxic effect of BP on various human bladder cancer cells. BP induced mitochondria-dependent apoptotic cell death, and inhibited the migration of the 4 bladder cancer cell lines. Interestingly, the 4 bladder cancer cell lines were isolated from different stages of bladder cancer. We thus speculated that BP might have therapeutic potential in treating bladder cancer of different stages. However, testing the therapeutic potential of BP in these stages of bladder cancers needs to be evaluated in different mouse models.
The mitochondria-dependent apoptotic cell death was evidenced by caspase-9 and caspase-3 cleavages in a time-dependent manner after BP treatments in these cell lines. Pretreatment of caspase-3 inhibitor (Z-DEVD-fmk) partly rescued the cells from BP cytotoxicity. The late-stage apoptosis was revealed by the TUNEL assay. The ability of BP in eliciting apoptotic cell death had also been documented in brain, lung and prostate cancer cells in previous studies [
9‐
11,
16].
Cell migration is essential for tumor metastasis, and metastasis is the most common fatal complication of all cancer patients [
25]. The migration of human bladder cancer cell lines was suppressed by BP in this study, which implicated the anti-metastasis effect of BP on bladder cancer cells. Several crucial steps such as loss of cellular adhesion, increased the motility and invasiveness, has been found to be associated with epithelial-mesenchymal transition (EMT) [
26]. The up-regulation of N-cadherin promotes motility, invasiveness and metastasis in tumor cells, whereas the loss of E-cadherin reduces the E-cadherin-mediated cell-cell adhesion and progress toward malignancy. Thus, these events play critical roles in EMT [
26]. Our results showed that BP-pretreatment significantly inhibited the migration and invasion of bladder cancer cells (Fig.
5a). In addition, BP suppressed the expression of N-cadherin but activated the expression of E-cadherin in the 4 bladder cancer cell lines (Fig.
5b). These results demonstrated the anti-metastasis effect of BP on bladder cancer cells. We thus speculated that BP might have therapeutic potential in treatment of bladder cancer of all stages.
Synergistic analysis of different anticancer agents is an important approach to determine the ratio and/or dose of drugs for clinical combination application [
27]. In previous studies, combination of cisplatin and other drugs could increase the cytotoxicity of chemotherapy in bladder cancer treatment [
28,
29]. In the present study, similar result of combined therapy was observed. Bladder cancer cells treated with BP demonstrated increased sensitivity to cisplatin, indicating that BP could be developed as a potential adjuvant cisplatin-based chemotherapy regimen. The mechanism of this synergistic effect includes drug inactivation, alterations in drug target, processing of drug-induced damage and evasion of apoptosis, which all needs to be clarified in the future [
30].
Acknowledgements
We thank to Ms. Hsin-Rong Wu, Department of Medical Research, Hualien Tzu Chi Hospital, for her assistance with animal experiments. This work was supported by grants from the Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taichung, Taiwan (TTCRD104-10, TTCRD104-19, TTCRD103-08, TTCRD103-12, TTCRD102-21).