Ginsenosides Synergize with Mitomycin C in Combating Human Non-Small Cell Lung Cancer by Repressing Rad51-Mediated DNA Repair
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Acta Pharmacologica Sinica (2018) 39: 449–458 © 2018 CPS and SIMM All rights reserved 1671-4083/18 www.nature.com/aps Article Ginsenosides synergize with mitomycin C in combating human non-small cell lung cancer by repressing Rad51-mediated DNA repair Min ZHAO, Dan-dan WANG, Yuan CHE, Meng-qiu WU, Qing-ran LI, Chang SHAO, Yun WANG, Li-juan CAO, Guang-ji WANG*, Hai-ping HAO* State Key Laboratory of Natural Medicines, Key Lab of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China The use of ginseng extract as an adjuvant for cancer treatment has been reported in both animal models and clinical applications, but its molecular mechanisms have not been fully elucidated. Mitomycin C (MMC), an anticancer antibiotic used as a first- or second- line regimen in the treatment for non-small cell lung carcinoma (NSCLC), causes serious adverse reactions when used alone. Here, by using both in vitro and in vivo experiments, we provide evidence for an optimal therapy for NSCLC with total ginsenosides extract (TGS), which significantly enhanced the MMC-induced cytotoxicity against NSCLC A549 and PC-9 cells in vitro when used in combination with relatively low concentrations of MMC. A NSCLC xenograft mouse model was used to confirm thein vivo synergistic effects of the combination of TGS with MMC. Further investigation revealed that TGS could significantly reverse MMC-induced S-phase cell cycle arrest and inhibit Rad51-mediated DNA damage repair, which was evidenced by the inhibitory effects of TGS on the levels of phospho- MEK1/2, phospho-ERK1/2 and Rad51 protein and the translocation of Rad51 from the cytoplasm to the nucleus in response to MMC. In summary, our results demonstrate that TGS could effectively enhance the cytotoxicity of MMC against NSCLC cells in vitro and in vivo, thereby revealing a novel adjuvant anticancer mechanism of TGS. Combined treatment with TGS and MMC can significantly lower the required concentration of MMC and can further reduce the risk of side effects, suggesting a better treatment option for NSCLC patients. Keywords: non-small cell lung carcinoma; total ginsenosides extract; mitomycin C; synergistic effect; cell cycle arrest; DNA damage repair; MEK1/2; ERK1/2; Rad51 Acta Pharmacologica Sinica (2018) 39: 449–458; doi: 10.1038/aps.2017.53; published online 24 Aug 2017 Introduction scription and replication[6]. Currently, MMC is being exten- Lung cancer is among the leading causes of cancer death sively used as a chemotherapeutic agent in the clinic for the among males in both developed and developing countries, and treatment of a variety of cancers, including stomach, breast, it has surpassed breast cancer among females in developed pancreas, colon and bladder cancer[7-9]. MMC has been formu- countries. Approximately 1.8 million new lung cancer cases lated to treat NSCLC as a single agent and can be combined were diagnosed in 2012, accounting for approximately 13% with other chemotherapy agents for advanced NSCLC treat- of total cancer diagnoses[1]. Non-small cell lung carcinoma ment in the form of a first- or second-line regimen. However, (NSCLC) accounts for 80% of lung cancers[2]. Chemotherapy it exhibits prolonged suppression of the bone marrow. There- is the main therapy for NSCLC patients, however, toxic side fore, the role of MMC in the treatment of NSCLC needs to be effects limit its clinical application to a great extent[3-5]. Mito- redefined[10, 11]. At present, NSCLC remains a threat to human mycin C (MMC) is an anticancer agent that causes intrastrand health, with an overall 5-year survival rate of only 18.2%[12]. DNA crosslinks (ICLs), which are among the most severe Many natural products have anticancer effects and can types of DNA damage and block DNA processes such as tran- enhance the cytotoxicity of chemotherapy agents[13, 14]. It is a common strategy to use combinations of natural products and chemotherapy agents as anticancer treatment to overcome *To whom correspondence should be addressed. drug resistance and reduce toxicity due to the safety and effi- E-mail [email protected] (Hai-ping HAO); [15, 16] [email protected] (Guang-ji WANG). cacy of natural products ; however, the underlying mecha- Received 2016-12-13 Accepted 2017-04-05 nisms of the antitumor effects of most natural products are www.nature.com/aps 450 Zhao M et al still unknown. were obtained from the American Type Culture Collection Ginseng (Panax ginseng C. A. Mey) has been widely used in (ATCC) (Maryland, USA) and cultured in RPMI-1640 medium Asian countries for thousands of years[17]. The effective com- supplemented with 10% fetal bovine serum, 100 U/mL peni- ponents of ginseng are multiple ginsenosides, including more cillin and 100 µg/mL streptomycin at 37 °C with 5% CO2. All than 100 types of protopanaxadiol (PPD) and protopanaxatriol reagents used in cell culture were from Life Technologies (PPT)-type ginsenosides[18]. The pharmacological activities (Shanghai, China). and pharmacokinetic behaviors of these ginsenosides have been identified in many studies[19-21]. Ginseng and Shenmai Cell proliferation assay injection (ginseng-derived Chinese medicine) are prescribed as Cells in 96-well plates at 80% confluence were treated with herbal medicines for cancer patients in China; however, their 0.25, 0.5 or 0.75 mg/mL TGS and 0.25, 0.5 or 0.75 µg/mL underlying adjuvant anticancer mechanisms are still elusive. MMC alone or in combination for 48 h. Cell viability was This study was designed with the aim of elucidating the measured using the CCK-8 assay according to the manufactur- signaling pathways associated with promoting cell death in er’s instructions. Absorbance at 450 nm was measured using a NSCLC by total ginsenosides extract (TGS) in combination BioTek Synergy H1 Hybrid Reader (Vermont, USA). with MMC. In brief, TGS significantly upregulated the anti- cancer effect of MMC in A549 and PC-9 cells in a synergistic Hoechst staining manner. The underlying mechanism of this synergistic effi- Cells in 6-well plates at 80% confluence were treated with cacy involved the MEK1/2-ERK1/2-Rad51 pathway, which is 0.5 or 1 mg/mL TGS and 0.5 or 1 µg/mL MMC alone or in responsible for the poor efficacy of long-term MMC treatment. combination for 24 h, after which the medium was discarded The synergistic efficacy of TGS combined with MMC was fur- and fresh medium containing 10 µg/mL Hoechst 33342 was ther confirmed in a xenograft modelin vivo, suggesting a novel added for further 30 min at 37 °C. Then, the medium was dis- strategy for combination therapy for NSCLC patients. carded, and the cells were gently washed three times with ice- cold PBS. The cells were subsequently observed with a Leica Materials and methods DMI3000B fluorescence microscope (Bensheim, Germany). Chemicals and reagents Total ginsenosides extract and ginsenoside monomers (purity siRNA transfection 98%) were purchased from the College of Chemistry of Jilin All transfections were performed using Lipofectamine iMAX University (Changchun, China). The ginsenosides content according to the manufacturer’s instructions. Cells were (Rb1 11.2%, Rb2 11.1%, Rc 10.5%, Rd 7.7%, Re 8.9%, Rf 0.9%, transfected with Rad51-specific or negative control siRNAs Rg1 3.3%, Rg2 1.4%, and Rh1 0.2%) in the total ginsenosides (GenePharma Inc, Shanghai, China) at a concentration of 20 extract was determined using a liquid chromatography-mass nmol/L for 48 h before the assays. The sequence of the siRNA spectrometry method validated in our laboratory[21]. TGS was duplex for RAD51 is as follows: sense strand: 5’-CCAC- dissolved in RPMI-1640 medium at a concentration of 100 CAGACCCAGCUCCUUUAUCAA-3’, antisense strand: mg/mL. Ginsenoside monomers were dissolved in DMSO at 5’-UUGAUAAAGGAGCUGGGUCUGGUGG-3’. a concentration of 100 mmol/L. Diol-type ginsenosides and triol-type ginsenosides were dissolved in DMSO at concentra- Apoptosis assay tions of 0.05 mg/mL and 0.025 mg/mL, respectively. MMC Cells were treated with 0.75, 1.25 and 1.5 mg/mL TGS and was purchased from Bomei Biotechnology (Hefei, China) 0.75, 1.25 and 1.5 µg/mL MMC for 24 h, washed with ice-cold and dissolved in DMSO at concentrations of 0.25, 0.5, 1 and PBS and fixed successively with 4% formaldehyde for 20 min 2 mg/mL. DMSO was purchased from Sigma-Aldrich (St at 4 °C and 75% ethanol for 2 h at -20 °C. The apoptotic cells Louis, MO, USA). The MEK1/2 inhibitor U0126-EtOH was were labeled using a FragELTM DNA Fragmentation Detection purchased from Selleckchem (Houston, USA). Lipofectamine Kit. The labeled cells (1×104) were analyzed using a BD Biosci- iMAX, Rad51 siRNA (VHS40454) and control siRNA (medium ences FACSCalibur flow cytometer (San Jose, CA, USA). GC) were purchased from Life Technologies (Shanghai, China). The FragELTM DNA Fragmentation Detection Kit Western blot analysis (QIA39) was purchased from Merck Millipore (Massachu- Cells were collected and lysed in ice-cold RIPA lysis buffer setts, USA). Cell Counting Kit 8 (CCK-8) was purchased from containing 2 mmol/L PMSF. The protein concentrations in Dojindo (Kumamoto, Japan). RPMI-1640 and Opti-MEM the lysates were quantified using a BCA Protein Assay Kit. medium were purchased from Gibco (California, USA). RIPA Cell lysates containing 60 µg of protein were subjected to SDS- lysis buffer, Hoechst 33342, Triton X-100, PMSF, BCA protein PAGE using 8%–12% gradient polyacrylamide gels (Bis-Tris assay kit, and nuclear and cytoplasmic protein extraction kit Midi Gel, Life Technologies, USA) and analyzed by immu- were purchased from Beyotime Biotechnology (Shanghai, noblotting with antibodies to the corresponding proteins.