Te-Fu Tsai

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.

Benzyl isothiocyanate induces protective autophagy in human prostate cancer cells via inhibition of mTOR signaling

Benzyl isothiocyanate (BITC) is a dietary chemopreventive agent that inhibits the growth of various human cancer cells by causing apoptotic cell death. In this study, we demonstrate that BITC not only induces apoptosis but also induces autophagy in human hormone-sensitive (Rv1) and -refractory (PC3) prostate cancer cells. In BITC-treated cells, the induction of autophagy was detected by monitoring the processing of an autophagy marker protein, microtubule-associated protein 1 light chain 3 (LC3), the aggregation of LC3 into granular structures and the formation of acidic organelles. Inhibition of autophagy using 3-methyladenine increased BITC-induced apoptosis, whereas the administration of caspase inhibitor suppressed BITC-induced cell death. Our data also showed that BITC inhibits mammalian target of rapamycin (mTOR) kinase activity in a dose-dependent manner. The expression of phospho-mTOR (Ser2481), an indicator of mTOR intrinsic catalytic activity, and phospho-UNC-51-like kinase 1 (Ser757), a direct substrate of mTOR, were decreased in BITC-treated cells. However, the increased expression of phospho-mTOR (Ser2448), phospho-AKT (Ser473) and antiapoptotic Bcl-2 were detected only in PC3 cells at later stages of BITC treatment. Collectively, our results show that BITC induces a protective autophagy response in Rv1 and PC3 cells through inhibition of the mTOR signaling pathway. Activation of the AKT survival pathway was only observed in PC3 cells, representing a resistance mechanism of advanced prostate cancer upon BITC treatment. These findings could potentially contribute to the beneficial effect of BITC in prostate cancer treatments.

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.

Benzyl isothiocyanate induces reactive oxygen species-initiated autophagy and apoptosis in human prostate cancer cells

Benzyl isothiocyanate (BITC) in cruciferous plants, which are part of the human diet, has been shown to induce apoptosis in various types of cancer. In this study, we show that BITC effectively suppresses the growth of cultured human prostate cancer cells (CRW-22Rv1 and PC3) by causing mitochondrial membrane potential loss, caspase 3/7 activation and DNA fragmentation. Furthermore, BITC induces ROS generation in these cells. The induction of apoptosis by BITC was significantly attenuated in the presence of N-acetylcysteine (NAC) and catalase (CAT), well-studied ROS scavengers. The induction of autophagy in BITC-treated cells were also diminished by the application of NAC or CAT. In addition, BITC-induced apoptosis and autophagy were both enhanced by the pretreatment of catalase inhibitor, 3-Amino-1,2,4-triazole (3-AT). Pretreatment with specific inhibitors of autophagy (3-methyladenine or bafilomycin A1) or apoptosis (Z-VAD-FMK) reduced BITC-induced autophagy and apoptosis, respectively, but did not abolish BITC-induced ROS generation. In conclusion, the present study provides evidences that BITC caused prostate cancer cell death was dependent on the ROS status, and clarified the mechanism underlying BITC-induced cell death, which involves the induction of ROS production, autophagy and apoptosis, and the relationship between these three important processes.

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.

Female Sexual Dysfunction: Physiology, Epidemiology, Classification, Evaluation and Treatment

Sexual health is defined by the World Health Organization as the integration of somatic, emotional, intellectual, and social aspects in ways that are positively enriching and that will enhance personality, communication, and love. The female sexual response is multifaceted and involves neurovascular, endocrine, and psychosocial factors. Optimal female sexual health comprises physical, mental, and emotional aspects, and these are the context in which a woman experiences desire, arousal, and orgasm. Sexual dysfunction in females is a complex and highly prevalent disease, and is commonly associated with the quality of life. Several factors influence female sexual function, including physiological and psychosocial factors. This article discusses models of female sexual response, defines and categorizes female sexual dysfunction, and identifies therapeutic modalities for female patients with sexual dysfunction.

Cisplatin induces protective autophagy through activation of BECN1 in human bladder cancer cells

Purpose Cisplatin-based chemotherapy is the first line treatment for several cancers including bladder cancer (BC). Autophagy induction has been implied to contribute to cisplatin resistance in ovarian cancer; and a high basal level of autophagy has been demonstrated in human bladder tumors. Therefore, it is reasonable to speculate that autophagy may account for the failure of cisplatin single treatment in BC. This study investigated whether cisplatin induces autophagy and the mechanism involved using human BC cell lines. Materials and methods Human BC cells (5637 and T24) were used in this study. Cell viability was detected using water soluble tetrazolium-8 reagents. Autophagy induction was detected by monitoring the levels of light chain 3 (LC3)-II and p62 by Western blot, LC3-positive puncta formation by immunofluorescence, and direct observation of the autophagolysosome (AL) formation by transmission electron microscopy. Inhibitors including bafilomycin A1 (Baf A1), chloroquine (CQ), and shRNA-based lentivirus against autophagy-related genes (ATG7 and ATG12) were utilized. Apoptosis level was detected by caspase 3/7 activity and DNA fragmentation. Results Cisplatin decreased cell viability and induced apoptosis of 5637 and T24 cells in a dose-and time-dependent manner. The increased LC3-II accumulation, p62 clearance, the number of LC3-positive puncta, and ALs in cisplatin-treated cells suggested that cisplatin indeed induces autophagy. Inhibition of cisplatin-induced autophagy using Baf A1, CQ, or ATG7/ATG12 shRNAs significantly enhanced cytotoxicity of cisplatin toward BC cells. These results indicated that cisplatin induced protective autophagy which may contribute to the development of cisplatin resistance and resulted in treatment failure. Mechanistically, upregulation of beclin-1 (BECN1) was detected in cisplatin-treated cells, and knockdown of BECN1 using shRNA attenuated cisplatin-induced autophagy and subsequently enhanced cisplatin-induced apoptosis. Conclusion Collectively, the study results indicated that cisplatin-induced autophagy is mediated by BECN1 in BC cells. Therefore, combinative treatment using cisplatin and autophagy inhibitors could potentially overcome cisplatin resistance related to autophagy induction.

Zoledronic Acid Elicits Proinflammatory Cytokine Profile in Osteolytic Prostate Cancer Cells

Zoledronic acid (ZA), a bisphosphonate used to prevent skeletal fractures in patients with cancers, was demonstrated to induce apoptosis in a number of cancer cells. Our previous study showed that ZA also induces autophagic cell death in metastatic prostate cancer cells. However, the clinical trials using ZA in the treatment of metastatic prostate cancer did not have a longer diseases-free period. Since most of ZA was attracted to the bone after administration, we hypothesized that local prostate cancer cells may evolve prosurvival pathways upon low concentration of ZA treatment. In this study, we investigated the inflammatory effects of ZA on osteolytic PC3 prostate cancer cell, since inflammation was reported to be related to cancer development and survival. Exposure of PC3 cells to various concentrations of ZA resulted in induction of apoptosis and autophagy. The expression of inflammatory biomarkers including interleukin 6 (IL-6), cyclooxygenase-2 (COX-2), and NF-κB was remarkably upregulated in response to ZA treatment in a dose- and time-dependent manner. The production of IL-6 was elevated upon ZA treatment. The antiapoptotic protein Bcl2 was increased with parallel increased level of IL-6. Our data suggest that treatment with low concentrations of ZA enhances the inflammatory profile and may serve as a prosurvival signaling pathway in PC3 cells.