Acta Pharmacol Sin 2007 Jul; 28 (7): 1057–1066 Full-length article P53-mediated cell cycle arrest and apoptosis through a caspase-3- independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells Qiao CUI1, Jing-hua YU1, Jin-nan WU1, Shin-ichi TASHIRO2, Satoshi ONODERA2, Mutsuhiko MINAMI3, Takashi IKEJIMA1,4 1China-Japan Research Institute of Medical Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China; 2Department of Clinical and Biomedical Sciences, Showa Pharmaceutical University, Tokyo 194-8543, Japan; 3Department of Immunology, Yokohama City University School of Medicine, Yokohama 235-0004, Japan Key words Abstract oridonin; MCF-7 cells; cell cycle arrest; Aim: To study the caspase-3-independent mechanisms in oridonin-induced MCF-7 apoptosis; p53; caspase-9; caspase-3; calpain; human breast cancer cell apoptosis in vitro. Methods: The viability of oridonin- mitochondrial pathway treated MCF-7 cells was measured by MTT (thiazole blue) assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleoso- 4 Correspondence to Prof Takashi IKEJIMA. mal DNA fragmentation was assayed by agarose gel electrophoresis. The apoptotic Phn/Fax 86-24-2384-4463. E-mail [email protected] ratio was determined by lactate dehydrogenase assay. Cell cycle alternation and mitochondrial membrane potential were measured by flow cytometric analysis. Received 2006-11-08 Bax, Bcl-2, caspase-3, caspase-9, heat shock protein (Hsp)90, p53, p-p53, p21, Poly Accepted 2007-01-30 (ADP-ribose) polymerase (PARP), and the inhibitor of caspase-activated DNase doi: 10.1111/j.1745-7254.2007.00588.x (ICAD) protein expressions were detected by Western blot analysis. Results: Oridonin inhibited cell growth in a time- and dose-dependent manner. Cell cycle was altered through the upregulation of p53 and p21 protein expressions. Pan- caspase inhibitor Z-VAD-fmk and calpain inhibitor Ⅱ both decreased cell death ratio. Nucleosomal DNA fragmentation and the downregulation of ∆ψmit were detected in oridonin-induced MCF-7 cell apoptosis, which was involved in a postmitochondrial caspase-9-dependent pathway. Decreased Bcl-2 and Hsp90 expression levels and increased Bax and p21 expression levels were positively correlated with elevated levels of phosphorylated p53 phosphorylation. Moreover, PARP was partially cleaved by calpain rather than by capase-3. Conclusion: DNA damage provoked alternations in the mitochondrial and caspase-9 pathways as well as p53-mediated cell cycle arrest, but was not related to caspase-3 activity in oridonin-induced MCF-7 cells. cell proliferation, as well as enhances the sensitivity of drug- Introduction resistant cancer cell lines[6,7]. Herbal medicine, Donglingcao (rabdosia rubescens), has Apoptosis is an essential and highly conserved mode of been traditionally used in China for the treatment of leukemia. cell death that is important for normal development, host Oridonin is a diterpenoid compound isolated from rabdosia defense, and the suppression of oncogenesis. Apoptosis rubescens. It has potent antitumor functions[1] and has been removes cancerous or virally-infected cells, and aberrant used for the treatment of human cancers, especially esoph- apoptosis is the major cause for tumor development and pro- ageal carcinoma[2]. This compound has been observed to gression[8]. Among the numerous proteins and genes prevent mutation, decrease Na+-pump transportation activ- involved, members of caspase family and the Bcl-2 family ity of cancer cells, and promote the efficiency of other anti- play pivotal roles in modulating apoptosis. Apoptosis is tumor agents[3–5]. It induces apoptosis to inhibit cancerous mediated by the activation of caspases, which amplify the ©2007 CPS and SIMM 1057 Cui Q et al Acta Pharmacologica Sinica ISSN 1671-4083 apoptotic signal and proteolytically process numerous cel- Manassas, VA, USA). The cells were cultured in RPMI-1640 lular target molecules with various functions[9]. Bcl-2 family medium (GIBCO,LA, NY, USA) supplemented with 10% FBS proteins are vital for the regulation of apoptosis by control- and 0.03% L-glutamine (GIBCO, USA) and maintained at 37 ling the mitochondrial membrane potential to release cyto- °C with 5% CO2 in a humidified atmosphere. chrome c and for activating caspase-9 and -3[10,11]. The bal- Cell viability assay The cytotoxic effect of oridonin on ance between pro-apoptotic proteins and anti-apoptotic pro- the MCF-7 cells was measured by MTT assay as described teins determines the fate of cells. in previous studies[16]. The cells were dispensed in 96-well, The DNA damage response, caused by a variety of flat bottom microtiter plates (NUNC, Roskilde, Denmark) at a stimuli, arrests the cell cycle to allow damage repair or direct density of 1×104 cells per well. After 24 h incubation, they cell apoptosis[12]. An imbalance between DNA damage and were treated with various concentrations of oridonin, fol- DNA repair activities may affect cell viability. After DNA lowed by 12, 24, 36, and 48 h cell culture. 20 µL MTT solu- 3 damage, the cell cycle is arrested at the transition from the G1 tion (5.0×10 mg/L) was added to each well 4 h before the end to S phase or from the G2 to M phase of the cell cycle. In- of incubation. The effects of Z-VAD-fmk and calpain inhibi- creasing evidence indicates a central role for p53 in mediat- tor II on oridonin treated MCF-7 cells were also determined ing cell cycle arrest or apoptosis[13,14]. by MTT assay, and both inhibitors were added into the cell In the present study, we demonstrated that activated p53 culture 1 h before oridonin administration, respectively. The contributed to oridonin-induced cell cycle arrest and apopto- resulting crystals were dissolved in DMSO. Absorbance sis, and mitochondrial alternations amplified the activation was measured with an ELISA reader (TECAN SPECTRA, of the caspase cascade; meanwhile, caspase-9, together with Wetzlar, Germany). The cytotoxic effect was expressed as a calpain rather than caspase-3, led to oridonin-induced apopto- relative percentage of inhibition calculated as follows: sis in human breast MCF-7 cells. Relative inhibition (%)=[(A 490 control - A 490 oridonin)/A 490 control]×100 Observation of morphological changes The MCF-7 cells 5 Materials and methods were plated in the wells of a 6-well plate at a density of 3×10 cells per well. After cultured for 24 h, the cells were treated Chemicals Oridonin was obtained from Kunming Insti- with 80 µmol/L oridonin and incubated for 12 or 24 h. The tute of Botany (The Chinese Academy of Sciences, Kunming, cellular morphological changes were observed using phase China), and the structure of oridonin was assigned by com- contrast microscopy (Leica, Nussloch, Germany). paring the chemical and spectral data (1H-NMR, 13C-HMR) Membrane leakage assay The integrity of the plasma with those reported in other published literature[15]. The pu- membrane of the MCF-7 cells was determined by monitoring rity of oridonin was measured by HPLC and determined to the cell lactate dehydrogenase (LDH) leakage, which was be more than 99%. Oridonin was dissolved in dimethyl sul- accomplished by following the rate of conversion of NADH foxide (DMSO) to make a stock solution. DMSO concentra- to NAD+. LDH released into the extracellular medium was tions were kept at below 0.05% in all the cell cultures and did expressed as a percentage of total LDH[17,18]. In brief, 1.5×106 not exert any detectable changes in cell growth or apoptosis. cells were cultured with oridonin for 0, 8, 16, 24, and 36 h. Fetal bovine serum (FBS) was purchased from TBD Biotech- Floating dead cells were collected from the culture medium nology Development (Tianjin, China); propidium iodide (PI), by centrifugation, and the LDH content from the pellets ly- Rhodamine 123, and RNase A were purchased from Sigma sed in 0.1% NP-40 for 15 min was used as an index of apoptotic Chemical (St Louis, MO, USA). Thiazolyl blue (MTT) was cell death (LDHp). The LDH released in the culture medium from Sino-American Biotechnology (Beijing, China); Z-VAD- [extracellular LDH (LDHe)] was used as an index of necrotic fmk and calpain inhibitor II were bought from Sigma Chemi- cell death. The LDH released from adherent and viable cells cal (St Louis, MO, USA); rabbit polyclonal antibodies against was expressed as intracellular LDH (LDHi). The substrate Bax, p53, p-p53, p21, caspase-9 and -3, PARP, heat shock reaction buffer of LDH was added. The A value at 490 nm of protein (Hsp)90, the inhibitor of caspase-activated DNase reaction for 1 and 5 min was assayed. LDH activity=(A5 min- (ICAD), mouse polyclonal antibodies against Bcl-2, and A1 min)/4. The percentage of apoptotic and necrotic cell death horseradish peroxidase-conjugated secondary antibody was calculated as follows: (goat-anti-rabbit or goat-anti-mouse) were purchased from % apoptosis=[LDHp/(LDHp+LDHe+LDHi)]×100 Santa Cruz Biotechnology (Santa Cruz, CA, USA). % necrosis=[LDHe/(LDHp+LDHe+LDHi)]×100 Cell culture The MCF-7 cell line (#CRL HTB-22) was Mitochondrial transmembrane potential alternation purchased from American Type Culture Collection (ATCC, Mitochondrial transmembrane potential (∆ψmit) alternation 1058 Http://www.chinaphar.com Cui Q et al was determined by Rhodamine 123 staining. For the fluores- aprotinin). The protein concentration was determined by cence observation, the MCF-7 cells were seeded into 6-well the Bio-Rad DC protein assay (Bio-Rad Laboratories, plates and mounted on the coverslips. For the flow cytometric Hercules, CA, USA). The protein lysates were separated by analysis, the cells were harvested by tripsin. After culture 12% SDS-PAGE and transferred to a nitrocellulose membrane. with oridonin for 12 h, the cells were removed from the cul- The membranes were soaked in blocking buffer (5% skimmed ture medium for staining.