Yi-Chia Lin

Zoledronic acid induces autophagic cell death in human prostate cancer cells

PURPOSE Bisphosphonates are potent inhibitors of bone resorption. In vitro studies show that zolendronic acid inhibits prostate cancer cell growth by activating apoptosis. We investigated whether zolendronic acid also inhibits prostate cancer cell growth by autophagy (type II programmed cell death). MATERIALS AND METHODS We investigated the induction of autophagy in the PC-3, DU-145, LNCaP and CRW22Rv1 cell lines upon zolendronic acid treatment. LC3-II protein formation was detected by Western blot. LC3-II incorporation into autophagosomes was detected by immunofluorescence staining. Acidic organelle formation was detected by acridine orange staining. Rescue experiments using an apoptosis inhibitor and/or an autophagy inhibitor were performed by MTT assay. RESULTS Autophagy induction was detected by formation of the LC3-II protein after exposure to 100 μM zolendronic acid. LC3-II and caspase-3 processing was detected 6 days after treatment. Acidic organelles were detectable by acridine orange staining and immunofluorescence showed round-up and condensed staining of LC3-II, suggesting autophagosome formation in the cytoplasm during autophagic cell death. Cell growth was rescued only by administering an apoptosis and autophagy inhibitor during zolendronic acid treatment, indicating that zolendronic acid induces prostate cancer death by apoptotic and autophagic cell death. CONCLUSIONS To our knowledge we report the first study showing that zolendronic acid markedly inhibits human prostate cancer cell growth through autophagic cell death. Zolendronic acid shows its anticancer activity via apoptosis and autophagy. These findings can potentially contribute to the beneficial use of zolendronic acid for prostate cancer treatment.

A survey of erectile dysfunction in Taiwan: use of the erection hardness score and quality of erection questionnaire.

INTRODUCTION There are currently no studies in the Asia-Pacific region using the erection hardness score (EHS) and Quality of Erection Questionnaire (QEQ) to assess erectile dysfunction (ED). AIMS To provide up-to-date data on the prevalence of ED in Taiwanese men and to validate the EHS and QEQ in this population. METHODS A representative sample of 1,060 men aged ≥ 30 years completed a telephone interview. ED status was confirmed via direct questioning and using the abridged five-item version of the 15-item International Index of Erectile Function (IIEF-5). Responses regarding EHS, QEQ, marital and sexual satisfaction, and attitude to treatment were also recorded. MAIN OUTCOME MEASURES IIEF, EHS, and QEQ. RESULTS The prevalence of ED, as defined by IIEF-5, was 27% among all respondents and 29% among those aged ≥ 40 years. Although, the prevalence of ED increased with age, men of all ages tended to underestimate their erectile problems. Among men who indicated that they did not have ED, 25% were found to have mild to moderate ED according to the IIEF-5 assessment. An EHS ≤ 3, indicating the presence of ED, was reported in 26% of men. The EHS was consistent with the QEQ: When the EHS was 4, the satisfaction of each domain of QEQ ranged from 85% to 90%. The QEQ score correlated well with the IIEF-5 score and significantly affected both sexual and marital satisfaction (P < 0.005). CONCLUSIONS These data indicate that EHS is a simple, practical tool for clinical use. QEQ scores appear to be independently associated with sexual and marital satisfaction, and may be of value in the assessment and monitoring of ED patients. While ED is a common health problem in Taiwan and the prevalence of ED increases with age, affected men lack awareness regarding the presence of erectile problems and the importance of initiating timely and effective treatment.

Chloroquine and hydroxychloroquine inhibit bladder cancer cell growth by targeting basal autophagy and enhancing apoptosis

Chloroquine (CQ) and hydroxychloroquine (HCQ), two antimalarial drugs, are suggested to have potential anticancer properties. in the present study, we investigated the effects of CQ and HCQ on cell growth of bladder cancer with emphasis on autophagy inhibition and apoptosis induction in vitro. The results showed that CQ and HCQ inhibited the proliferation of multiple human bladder cell lines (including RT4, 5637, and T24) in a time‐ and dose‐dependent fashion, especially in advanced bladder cancer cell lines (5637 and T24) compared to immortalized uroepithelial cells (SV‐Huc‐1) or other reference cancer cell lines (PC3 and MCF‐7). We found that 24‐hour treatment of CQ or HCQ significantly decreased the clonogenic formation in 5637 and T24 cells compared to SV‐Huc‐1. As human bladder cancer tumor exhibits high basal level of autophagic activities, we detected the autophagic flux in cells treated with CQ and HCQ, showing an alternation in LC3 flux in CQ‐ or HCQ‐treated cells. Moreover, bladder cancer cells treated with CQ and HCQ underwent apoptosis, resulting in increased caspase 3/7 activities, increased level of cleaved poly(ADP‐ribose) polymerase (PARP), caspase 3, and DNA fragmentation. Given these results, targeting autophagy with CQ and HCQ represents an effective cancer therapeutic strategy against human bladder cancer.

Low-dose testosterone treatment decreases oxidative damage in TM3 Leydig cells

Testosterone replacement therapy has benefits for aging men and those with hypogonadism. However, the effects of exogenous testosterone on Leydig cells are still unclear and need to be clarified. In this report, we demonstrate that testosterone supplementation can reduce oxidative damage in Leydig cells. The TM3 Leydig cell line was used as an in vitro cell model in this study. Cytoprotective effects were identified with 100-nmol l⁻¹ testosterone treatment, but cytotoxic effects were found with ≥ 500-nmol l⁻¹ testosterone supplementation. Significantly reduced reactive oxygen species (ROS) generation, lipid peroxide contents and hypoxia induction factor (HIF)-1α stabilization and activation were found with 100-nmol l⁻¹ testosterone treatment. There was a 1.72-fold increase in ROS generation in the 500-nmol l⁻¹ compared to the 100-nmol l⁻¹ testosterone treatment. A 1.58-fold increase in steroidogenic acute regulatory protein (StAR) expression was found in 50-nmol l⁻¹ testosterone-treated cells (P < 0.01). Chemically induced hypoxia was attenuated by testosterone supplementation. Leydig cells treated with low-dose testosterone supplementation showed cytoprotection by decreasing ROS and lipid peroxides, increasing StAR expression and relieving hypoxia stress as demonstrated by HIF-1α stabilization. Increased oxidative damage was found with ≥ 500-nmol l⁻¹ testosterone manipulation. The mechanism governing the differential dose effects of testosterone on Leydig cells needs further investigation in order to shed light on testosterone replacement therapy.

Trimodality bladder-sparing approach without neoadjuvant chemotherapy for node-negative localized muscle-invasive urinary bladder cancer resulted in comparable cystectomy-free survival

BackgroundTo retrospectively review the efficacy and organ preservation experience for muscle-invasive bladder cancer by trimodality therapy at our institution.MethodsBetween July 2004 and February 2012, seventy patients (M/F = 55/15; median age = 69 years) of lymph node negative localized muscle-invasive bladder cancer were treated primarily with trimodality approach including transurethral resection of bladder tumor (TURBT) prior to combined chemotherapy and radiotherapy (CCRT). Radiotherapy consisted of initial large field size irradiation with 3D conformal technique (3D-CRT), followed by cone-down tumor bed boost with intensity modulated radiotherapy (IMRT) technique. The median total doses delivered to bladder tumor bed and whole bladder were 59.4Gy and 40.0Gy, respectively. No patient received neoadjuvant chemotherapy (NAC). Weekly cisplatin was administered during radiotherapy. Toxicity was scored according to the RTOG criteria. Tumor response was evaluated both cystoscopically and radiographically 3 months after treatment.ResultsThe numbers of patients with T2, T3 and T4 lesions were 41, 16 and 13, respectively. Overall survival (OS) and progression-free survival (PFS) at 2 and 5 year were 65.7%, 51.9% and 50.8%, 39.9%, respectively, after a median follow-up time of 24 months. Local-regional control and distant metastasis free survival at 2 year were 69.8% and 73.5%, respectively. Complete response (CR) rate assessed three month after CCRT was 78.1%. Ten patients (20%) had local recurrence after initial CR (n = 50), 3 of them were superficial recurrence. One patient underwent radical cystectomy after recurrence. The overall 5-year bladder intact survival was 49.0% (95% CI, 35.5% to 62.5%). Acute toxicities were limited to grade 1-2. One patient developed late grade 3 GU toxicity.ConclusionsOur result suggested that trimodality bladder-sparing approach without NAC or dose-intensification could be well-tolerated with a high CR rate and bladder preserving rate for muscle-invasive bladder cancer.

Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells

Background Mammalian target of rapamycin (mTOR), involved in PI3K/AKT/mTOR pathway, is known to play a central role in regulating the growth of cancer cells. The PI3K/AKT/mTOR pathway enhances tumor survival and proliferation through suppressing autophagy, which sustains energy homeostasis by collecting and recycling cellular components under stress conditions. Conversely, inhibitors of the mTOR pathway such as RAD001 induce autophagy, leading to promotion of tumor survival and limited antitumor efficacy. We thus hypothesized that the use of autophagy inhibitor in combination with mTOR inhibition improves the cytotoxicity of mTOR inhibitors in bladder cancer. Materials and methods The cytotoxicity of RT4, 5637, HT1376, and T24 human bladder cancer cells treated with RAD001 alone or combined with autophagy inhibitors (3-methyladenine (3-MA), bafilomycin A1 (Baf A1), chloroquine, or hydroxychloroquine) was assessed using the WST-8 cell viability kit. The autophagy status in cells was analyzed by the detection of microtubule-associated light chain 3 form II (LC3-II), using immunofluorescent staining and Western blot. Acidic vesicular organelle (AVO) formation in treated cells was determined by acridine orange vital staining. Inhibition of mTOR pathway by RAD001 was monitored by using a homemade quantitative polymerase chain reaction gene array, while phospho-mTOR was detected using Western blot. Induced apoptosis was determined by measurement of caspase 3/7 activity and DNA fragmentation in cells after treatment. Results Advanced bladder cancer cells (5637, HT1376, and T24) were more resistant to RAD001 than RT4. Autophagy flux detected by the expression of LC3-II showed RAD001-induced autophagy. AVO formation was detected in cells treated with RAD001 and was inhibited by the addition of 3-MA or Baf A1. Cotreatment of RAD001 with autophagy inhibitors further reduced cell viability and induced apoptosis in bladder cancer cells. Conclusion Our results indicate that simultaneous inhibition of the mTOR and autophagy pathway significantly enhances apoptosis, and it is suggested to be a new therapeutic paradigm for the treatment of bladder cancer.

Induction of testicular damage by daily methamphetamine administration in rats

Methamphetamine (METH)-induced brain damage and apoptosis within the central nervous system are well documented. This study was conducted to investigate the toxic effects of daily METH administration on the testes in a rat model. Male Sprague-Dawley rats (5 weeks old, ~100 g, n = 64) were divided into two groups and treated with vehicle (saline, control) or METH (10 mg/kg) for 15, 30, 60 and 90 days. The results showed that daily administration of METH decreased the body, testicular and epididymis weights as well as the serum levels of total testosterone. The increased apoptotic index (Bad/Bcl2 expression ratio) and levels of cleaved caspase-3 indicated that apoptosis had occurred in the testes of the METH-treated rats. The oxidative stress levels increased as the reduced and oxidized glutathione (GSH/GSSG) ratio decreased. The overall sperm counts decreased at 15 and 90 days, where- as morphologically abnormal sperm counts increased at 30, 60 and 90 days in the METH-treated rats. This study demonstrates that daily exposure to METH significantly reduced the number and quality of sperm in rats. The underlying pathophysiological mechanisms likely include the reduction of serum testosterone levels and the increase of oxidative stress and apoptosis in the rat testes.

Acridine orange exhibits photodamage in human bladder cancer cells under blue light exposure

Human bladder cancer (BC) cells exhibit a high basal level of autophagic activity with accumulation of acridine-orange(AO)-stained acidic vesicular organelles. The rapid AO relocalization was observed in treated BC cells under blue-light emission. To investigate the cytotoxic effects of AO on human BC cell lines under blue-light exposure, human immortalized uroepithelial (SV-Huc-1) and BC cell lines (5637 and T24) were treated with indicated concentrations of AO or blue-light exposure alone and in combination. The cell viability was then determined using WST-1, time-lapse imaging with a Cytosmart System and continuous quantification with a multi-mode image-based reader. Treatment of AO or blue-light exposure alone did not cause a significant loss of viability in BC cells. However, AO exhibited a dose-dependent increment of cytotoxicity toward BC cells under blue-light exposure. Furthermore, the tumor formation of BC cells with treatment was significantly reduced when evaluated in a mouse xenograft model. The photodamage caused by AO was nearly neglected in SV-Huc-1 cells, suggesting a differential effect of this treatment between cancer and normal cells. In summary, AO, as a photosensitizer, disrupts acidic organelles and induces cancer cell death in BC cells under blue-light irradiation. Our findings may serve as a novel therapeutic strategy against human BC.

Complications after transrectal ultrasound-guided prostate biopsy: A 10-year experience in a single institution

Background: Transrectal ultrasound-guided prostate (TRUS-P) biopsy is the standard diagnostic procedure for patients with suspected prostate cancer. However, infectious complications can occur. Purpose: To analyze the patients from a single institution who were hospitalized for infectious complications after TRUS-P biopsy. Materials and Methods: From 2003 to 2012, 985 TRUS-P biopsy procedures were performed in a medical center in Northern Taiwan; among these, 28 patients were admitted for infectious complications following the procedure. A retrospective review of the medical records was performed, and data regarding the demographics, details of infectious complications, and hospital course of these patients were collected. Results: The median age of the patients was 62 (27–82) years; the comorbidity rate was 57.1% (16/28); the admission rate was 2.8% (28/985). The most common causative pathogen was Escherichia coli. More than half of the cultured E. coli were not resistant to fluoroquinolones. The median hospital stay was 6 (3–16) days, and one patient was admitted to the intensive care unit. No mortality was observed in this study. Although the number of biopsies increased, no obvious increasing trend in the number of hospitalizations was noted in recent years. Conclusion: TRUS-P biopsy can be safely performed, and no obvious increasing trend in infectious complications and resistant strains has been observed in recent years.