THE ANATOMICAL RECORD 295:417–422 (2012) Study on the Autophagy of Prostate Cancer PC-3 Cells Induced by Oridonin LI-HONG YE,* WANG-JIAN LI, XIAO-QIANG JIANG, YONG-LIANG CHEN, SHUI-XIANG TAO, WEI-LIANG QIAN, AND JIAN-SONG HE Department of Urology Surgery, Shaoxing County Central Hospital, Shaoxing 312030, China ABSTRACT To investigate the mechanism of oridonin (ORI)-induced autophagy in prostate cancer PC-3 cells, PC-3 cells cultured in vitro were treated with ORI, and the inhibitory ratio of ORI on PC-3 cells was assayed by 3-4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. The ultrastructural changes of the cells were observed under light microscope, scanning electron microscope (SEM), and transmission electron microscope (TEM). Acridine orange (AO) staining was used to observe the acidic vesicular organelles (AVOs). The level of autophagy-related proteins, MAP1-LC3, was detected by Western Blot, and RT-PCR was used to detect the level of mRNA of beclin 1. After ORI treatment, the proliferation of PC-3 cells was inhibited significantly in a concentration and time-dependent manner. SEM examination revealed cellular shrinkage and disappearance of surface microvilli in ORI-treated cells. Under TEM examination, the nuclei exhibited chromatin condensation and the appearance of a large number of autophagosomes with double-membrane structure in cytoplasm. AO staining showed the existence of AVOs. The expression of LC3 and the mRNA level of beclin 1 was increased by ORI. Furthermore, autophagy inhibitor 3-methyladenine reversed the increase of beclin 1 mRNA. The growth of PC-3 cells was inhibited, and autophagy was induced by ORI, indicating ORI may have a potential antitumor effect. C 2011 Wiley Periodicals, Inc. Anat Rec, 295:417–422, 2012. V Key words: oridonin; prostate cancer; autophagy; beclin 1 Oridonin (ORI), a diterpenoid extracted from Rabdosia rubescens, has been proven to have effective antitumor activity, in addition to its potential as an anti-inflammatory and antibacterial drug. It has been reported that ORI could significantly inhibited cell growth in ehrlich ascites carcinoma (EAC), sarcoma S180, mouse Leukemia P388 and L1210, and other transplantable tumors (Fujita et al., 1981). Meanwhile, ORI also has been widely used for the treatment of solid tumors, such as esophageal and gastric cancers, and has achieved significant clinical benefit (Han et al., 2003). Therefore, ORI can potentially be very effective antitumor drug. Autophagy is a ubiquitous process of nonapoptotic form of programmed cell death (PCD). In this process, formation of double-membrane vesicular structures, containing damaged organelles and part of cytosol, is caused. The vesicles subsequently delivered their contents to lysosome for degradation. Therefore, autophagy is a process of self-digestion and recycling of cellular C 2011 WILEY PERIODICALS, INC. V constituents, which helps cells to survive longer from starve. However, excessive autophagy can lead to autophagic cell death, which is termed type II PCD. In recent years, it has been reported that ORI could induce autophagy in HeLa, A431, and several other cancer cell lines and delay the occurrence of apoptosis (Cui et al., 2006; Cui et al., 2007a). However, the involvement of ORI-induced autophagy in prostate cancer has not been reported. In this study, we investigated the mechanism of ORI-induced autophagy in prostate cancer PC-3 cells in order to provide Grant sponsor: Zhejiang TCM Science Foundation; Grant number: 08029. *Correspondence to: Dr. Li-Hong Ye, Department of Urology Surgery, Shaoxing County Central Hospital, Shaoxing 312030, China. Fax: þ086-84122298. E-mail: firstname.lastname@example.org DOI 10.1002/ar.21528 Published online 20 December 2011 in Wiley Online Library (wileyonlinelibrary.com). 418 YE ET AL. experimental evidence for the development of new antitumor natural medicine. MATERIAL AND METHODS Reagents ORI purchased from the Wuhan Botanical Garden, China, was extracted as described previously by Jian et al. (2007) and detected by RP-HPCL (=98%). 3-4,5Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 3-methyladenine (3-MA) were purchased from SIGMA. F-12 medium was purchased from GIBCO, and fetal bovine serum (FBS) was obtained from ExCell Biology. Acridine orange (AO) was purchased from Fluka. The mouse monoclonal GAPDH antibody and goat LC3 antibody were obtained from Santa Cruz. Reverse transcription reagents and primers were purchased from Invitrogen. Cell Culture Prostate cancer PC-3 cells, preserved in our laboratory and obtained from ATCC, were seeded in F-12 medium (10% FBS, 100 U/L penicillin and streptomycin) and maintained at 37 C in a humidified atmosphere of 5% CO2. PC-3 cells were incubated with increasing concentrations of ORI drug. The control group was treated with equal-volume DMSO instead of drug. MTT Assay During the logarithmic growth phase, cells were digested with trypsin, suspended in F-12 medium (5 104 cells/mL), then cultured in 96-well plates (100 lL/ well). After 24 hr, the original media was removed and replaced with new culture medium containing various concentrations of ORI (5, 10, 15, 25, 50, and 100 lmol/ L). Each treatment was performed in triplicate and treated for 24 or 48 hr. Then the culture medium was replaced with 100 lL DMSO. After shaking on a rocker for 30 min, OD values were read on 128-C enzymelinked immunosorbent assay reader (CliniBio) at 492 nm wavelength, and the inhibition rate was calculated. Scanning Electron Microscopy PC-3 cells in the logarithmic phase were inoculated on sterilized cover slips covered with 1% paraformaldehyde in 24-well plates. After culture for 48 hr, cells were treated with 15 lmol/L ORI for 24 hr, washed with PBS two times, fixed in 2.5% glutaraldehyde at 4 C for 2 hr, and then washed again. Subsequently, it was dehydrated with 50%, 70%, 80%, 90%, and 100% ethanol gradient followed by further dehydration in 100% acetone for 15 min and finally washed in isoamyl acetate for 15 min. Critical point-dried in CO2, sputter-coated with gold and examined under scanning electron microscope (SEM; STEREOSCAN 260) with an accelerating voltage of 20 kV. Transmission Electron Microscopy PC-3 cells cultured in 100-mL flasks were treated with DMSO or ORI. After that, cells were collected, washed with PBS, fixed in 2.5% glutaraldehyde at 4 C for 2 hr, and then washed again twice with PBS. The material was dehydrated in a graded series of ethanol Fig. 1. Growth curves of PC-3 cells detected by MTT assay. A: ORI inhibited the proliferation of PC-3 cells. Cells were treated with different concentrations of ORI (5, 10, 15, 25, 50, and 100 lmol/L) for 24 and 48 hr, and then detected. B: Comparison of the effects of DMSO and ORI on cell proliferation. (50%, 70%, 80%, 90%, and 100%) and acetone for 15 min each and embedded in Epon 812. Ultrathin sections were stained with uranyl and lead acetates and examined under transmission electron microscope (TEM; Philips Tecnai 10) at 80 kV. AO Staining PC-3 cells were seeded on sterilized cover slips covered with 1% paraformaldehyde in 24-well plates (50,000 cells per well). After cultured for 24 hr, cells were treated with ORI for 24 hr, washed with PBS at 4 C twice, and stained with AO dye for 10 min in dark. Subsequently, cells were washed again and examined under a fluorescence microscope. Western Blot Cells treated with different ORI concentrations were collected and washed with PBS, and protein was extracted from lysates (Lowry method). Bicinchoninic acid protein assay kit (Pierce) was used to determine protein concentration. Equal denatured protein (50 lg) was separated by 12% SDS-PAGE electrophoresis and transferred to PVDF membrane. After blocked for 4 hr with nonfat dry milk, the blots were incubated with a dilution of primary antibody overnight and respective secondary antibody for 2 hr at room temperature. Immunoblots were developed by enhanced chemiluminescence. THE AUTOPHAGY OF PROSTATE CANCER PC-3 CELLS INDUCED BY ORIDONIN 419 Fig. 2. Morphological changes of ORI-treated PC-3 cells. Cells were treated with 20 lmol/L ORI for 24 hr and observed under Optical microscope (A, 200), or scanning electron microscope (B). RT-PCR Cells cultured in six-well plates were treated with ORI and lysed in 1 mL TRIzol for 5 min at room temperature to extract total RNA. Then RNA was reverse transcribed into cDNA and amplified using PCR. Amplification conditions were as follows: 94 C for 5 min; 94 C for 1 min; 52 C for 1 min; 72 C for 1 min; 30 cycles; 72 C for 10 min. The primer sequences were as follows: • GAPDH forward: CCAGCCGAGCCACATCGCTC, • GAPDH reverse: ATGAGCCCCAGCCTTCTCCAT (588 bp); • beclin 1 forward: TGGATCACCCACTCTGTGAG, • beclin 1 reversed: TTATTGGCCAGAGCATGGAG3 (569 bp). 39.95 6 3.05% after 24 hr, and reached 51.28 6 2.91% after 48 hr. When the concentration of ORI was increased to 25 lmol/L, the inhibition rate was increased to 67.94 6 4.78% (Fig. 1A). When compared with ORI, equal concentrations of DMSO solvent mildly promote proliferation of PC-3 cells (Fig. 1B). Under light microscope, cells treated with ORI at the concentration of 20 lmol/L (IC50 for 24 hr) for 24 hr were observed. We found cells reduced in number and appeared shrunken and rounded (Fig. 2A). Under SEM, drug-treated fusiform cells were found to became round, and microvilli on cell surface disappeared (Fig. 2B). The results suggested that ORI could inhibit prostate cancer PC-3 cell proliferation significantly in a concentration- and timedependent manner. RESULTS ORI Inhibited the Proliferation of PC-3 Cells Formation of Autophagy in ORI-treated PC-3 Cells Different concentrations of ORI (5, 10, 15, 25, 50, and 100 lmol/L) were treated PC-3 cells for 24 or 48 hr, and then cell viability was assayed by MTT. At the concentration of 15 lmol/L, the inhibition rate of ORI was Under TEM, control cells presented abundant of microvilli on cell surface, normal nucleo and nuclear-tocytoplasmic (N/C) ratio was 1:1 (Fig. 3A). PC-3 cells treated with ORI at the concentration of 20 lmol/L for 420 YE ET AL. Fig. 3. Ultrastructural changes of ORI-treated PC-3 cells observed under TEM. Cells were treated with DMSO or 20 lmol/L ORI for 24 hr, and then observed. (A) Normal control; (B) Drug-treated cells; (C) Autophagosome with double membrane (arrow). Fig. 4. Detection of acidic vesicular organelles (AVOs) with AO staining. Cells were treated with DMSO or 20 lmol/L ORI for 24 hr, and then stained and observed. Red fluorescent spots indicated AVOs. (A) DMSO control group; (B) Drug-treated group (200). 24 hr appeared to have shrunken cell nuclei with the integration of crescent-shaped chromatin, and the N/C ratio increased (Fig. 3B). Additionally, we observed existence of a large number of autophagic bodies with double membrane, containing portions of the cytoplasm and damaged organelles (Fig. 3C). By vital AO staining, control cells showed homogeneous green fluorescence in cytoplasm, while there were red fluorescent spots appeared in ORI-treated cells (Fig. 4), which suggested that ORI treatment caused formation of acidic vesicular organelles (AVOs) in PC-3 cells. Western blot showed that the expression of LC3-I protein increased in the PC-3 cells treated with low concentrations of ORI (10 lmol/L), but LC3-II protein was not detected. When the concentration of ORI was increased to 20 and 40 lmol/L, the expression of LC3-I protein decreased, while the expression of LC3-II protein was increased significantly (Fig. 5). This suggested that ORI increased the expression of LC3 protein and promoted the modification of LC3-I to LC3-II. Our results showed that ORI can induce autophagy in prostate cancer PC-3 cells. Elevation of Beclin 1 mRNA Level Was Involved in ORI-Induced Autophagy RT-PCR results showed that with the concentration of ORI increased, the beclin 1 mRNA levels increased in a concentration-dependent manner (Fig. 6A). Treatment of autophagy inhibitor 3-MA did not change beclin 1 mRNA levels in PC-3 cells. However, combined treatment of ORI and 3-MA could prevent ORI-induced upregulation of beclin 1 mRNA levels (Fig. 6B). The THE AUTOPHAGY OF PROSTATE CANCER PC-3 CELLS INDUCED BY ORIDONIN 421 Fig. 5. The expression levels of LC3 protein examined by Western Blotting. The expression of LC3 protein was increased by ORI. Cells were treated with DMSO or different concentrations of ORI (20 or 40 lmol/L). results suggest that ORI promotes beclin 1 gene expression at the transcriptional level, and this gene is involved in the regulation of ORI-induced autophagy. DISCUSSION ORI has varied pharmacologic activities, such as antiinflammatory, antibacterial, and antitumor activities. It can inhibit proliferation of tumor cells in breast cancer, colon cancer, and liver cancer and induce apoptosis in various cancer cell lines. ORI has been reported to block breast cancer cell line MCF-7 in S phase (Cui et al., 2007b) and colon cancer cell line HT29 in G2/M phase (Zhu et al., 2007) and induce characteristic apoptotic changes in hepatocellular carcinoma cell line BEL-7402 (Zhang et al., 2006), breast cancer cell line MCF-7 (Cui et al., 2007b) and human cervical cancer cells HeLa (Zhang et al., 2004). Ikezoe et al. (2003) found that ORI can inhibit androgen-dependent prostate cancer cell line LNCaP proliferation and block cells in the G0/G1 phase by upregulating p21WAF1 protein in a p53-dependent manner. However, the role of ORI in androgen-independent prostate cancer cell line PC-3 has not been reported. Therefore, we investigated the potential application of ORI in prostate cancer treatment. ORI was found to inhibit prostate cancer PC-3 cells significantly in a concentration- and time-dependent manner. We further studied the mechanisms of ORI-induced autophagy in PC-3 cells. After ORI treatment, there were a large number of autophagosomes and pre-autophagosomal structure with double-membrane in cytoplasm, associating with formation of AVOs, and conversion of LC3-I protein to LC3-II. ORI promoted transcription of beclin 1, which can be reversed by autophagy inhibitor 3-MA. Cui et al. (2006) found ORI-induced apoptosis, accompanied by the occurrence of autophagy at the same time in HeLa cells treated with ORI, indicating ORI had autophagy-inducing activity. In this study, we observed exist of autophagosome in ORI-treated cells for the first time. The occurrence of autophagosomes is a gold standard to determine autophagy. Therefore, our results provide strong evidence for autophagy-inducing activity of ORI. Autophagosome further fuses with lysosome to form autolysosome that is one kind of AVO structures. Because of this, formation of AVOs is another important indicator of autophagy and has been reported to be induced by various autophagic inducer, including radiation or ceramide (Daido et al., 2004; Paglin et al., 2001). Fig. 6. ORI-induced transcription of the beclin 1 gene in PC-3 cells. A: The level of beclin 1 mRNA was upregulated in ORI-treated cells. Cells were treated with DMSO or different concentrations of ORI for 24 hr, and the level of mRNA was detected by RT-PCR. B: Inhibitor of autophagy 3-MA blocked increase of mRNA transcription. Cells were treated by ORI (20 lmol/L) with or without 3-MA (5 mmol/L). Therefore, we determined the effect of ORI on the formation of AVOs in PC-3 cells by lysosomotropic agent AO staining and fluorescence microscopy. AO is a weak base that will accumulate in acidic compartments and form red fluorescence aggregates. MAP-LC3, the mammalian homolog of yeast Atg8, is an important marker of autophagy. LC3-I, evenly distributed throughout the cytoplasm, can be ubiquitin-like modified to LC3-II during autophagy, which associated with autophagosome membrane. LC3-II was further degraded by lysosomal hydrolases in lysosome. Therefore, LC3-I/ LC3-II ratio or LC3-II levels indicate the level of autophagy. Our research suggested that low concentrations of ORIinduced initial occurrence of autophagy but did not form mature autophagosomes. When concentration of ORI was increased, LC3-I/ LC3-II ratio decreased, indicating mature autophagosomes was formed. Thus, ORI-induced autophagy in PC-3 cells in a concentration-dependent manner. Autophagic phenomenon was first observed in liver cells of glucagon-treated mice by Ashford et al. (1962). As a cell survival mechanism, autophagy plays important roles in physiological clearing cell process, such as clearing damaged organelles. Nevertheless, excessive activation of autophagy can lead to cell death. It has been reported that autophagy was involved in tumor development. In early stage, autophagy inhibited tumor development (Gozuacik and Kimchi, 2004). While in advanced stages, autophagy helped cells to resist starvation and promoted cell growth (Cuervo, 2004). In addition, autophagy may protect several cancer cells from radiation damage. Because of its two-way effect in tumor development, induction or inhibition of autophagy determine the efficacy of anticancer drugs. ORI has been demonstrated to induce autophagy in human breast 422 YE ET AL. cancer MCF-7 cells and mouse fibroblast L929 cells. Furthermore, inhibition of autophagy in these two models could upregulate apoptosis (Cui et al., 2007a; Cheng et al., 2008). Our study found that induction of autophagy may be possible mechanism of ORI antiprostate cancer activity. Beclin 1 is the mammalian homolog of yeast autophagy protein Atg6 and plays an important role in formation of autophagy. It is involved in vacuolar transport via the cytoplasm-to-vacuole targeting (Cvt) pathway and the vacuolar protein-sorting (Vps) pathway. High-frequency monoallelic deletion of beclin 1 has been reported in human breast cancer, ovarian cancer, and prostate cancer. In MCF-7 cells, expression of beclin 1 protein was so low that it almost failed to detect, whereas stable tranfection of beclin 1 promoted autophagy in MCF-7 cells and reduced tumorigenic ability (Zhang et al., 1999). Broadspectrum caspase inhibitor zVAD has been demonstrated to inhibit activity of caspase-8 and induce autophagic cell death, in which Atg7 and beclin 1 genes played an essential role (Yu et al., 2004). Thus, beclin 1 may be a crossed protein in regulation of autophagy and apoptosis. Induction of autophagy may be the possible mechanism of several antitumor drugs, such as tamoxifen widely used in breast cancer therapy. Mediated by ceramide, tamoxifen could upregulate expression of Beclin 1 and activate cell autophagy (Scarlatti et al., 2004). ORI has been reported to increase expression of Beclin 1 in HeLa cells through Ras, JNK, and p38 regulation, which could be reversed by autophagy inhibitor 3-MA (Cui et al., 2007c). In our experiment, ORI promoted mRNA synthesis of beclin 1 gene, suggesting that ORI regulate beclin 1 expression at the transcriptional level. In addition, ORI treatment could decrease expression of antiapoptotic proteins Bcl-2, Bcl-xL, and promote the release of mitochondrial cytochrome C (Li et al., 2007; Liu et al., 2006). By interfering formation of Beclin-Ptdlns 3-kinase complex, Bcl-2 could inhibit activity of PtdIns 3-kinase and beclin 1-dependent autophagy (Cao and Klionsky, 2007). 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