|Home | About | Journals | Submit | Contact Us | Français|
Chemoresistance to anticancer drugs is a major obstacle in the efforts to develop a successful treatment strategy for esophageal squamous carcinoma (ESCC). Thus, the exploration of new drugs and treatment strategies for combating resistance are of utmost importance. In this study, we investigated the antitumor drug resistance activity of Jesridonin, a new ent-kaurene diterpenoid, and its possible mechanisms of action using the resistant cancer cell line, EC109/Taxol. MTT assay revealed that Jesridonin had similar IC50 values against EC109 paclitaxel-sensitive cells and drug-resistant EC109/Taxol cells in vitro. In mice, Jesridonin effectively prevented the growth of EC109/Taxol tumor xenografts without exerting any significant toxicity. In addition, Jesridonin significantly inhibited the proliferation of EC109/Taxol cells, induced apoptosis and arrested the cell cycle at the G2/M phase. Furthermore, western blot analysis revealed that Jesridonin upregulated the expression of p53, p53 upregulated modulator of apoptosis (PUMA), cleaved-caspase-9 and cleaved-caspase-3 in EC109/Taxol cells, and downregulated the expression of procaspase-3, procaspase-9 and Bcl-2 in the EC109/Taxol cells in a concentration-dependent manner. Overall, our results demonstrate that Jesridonin may have potential for use in the treatment of paclitaxel-resistant ESCC. The data of the present study may lead to the development of novel treatment strategies for paclitaxel-resistant tumors.
Chemotherapy is one of the main therapeutic strategies in the treatment of cancer. However, the success of chemotherapy depends on the sensitivity of the tumor to the anti-neoplastic agent (1). Cancer cells can develop resistance to the anticancer drugs used during cancer therapy (2). The drug resistance properties of the tumor greatly limit the effectiveness of chemotherapeutic drugs, resulting in the failure of chemotherapy and the recurrence of the tumor. Furthermore, cancer cells exposed to one drug may exhibit cross-resistance to other drugs that have different structures and functions. This phenomenon is known as multidrug resistance (MDR) and can be intrinsic or acquired following chemotherapy using a cytotoxic agent (3,4). Resistance to anticancer drugs is a major obstacle for successful chemotherapy. Thus, the exploration of new drugs and strategies with which to combat drug resistance is of utmost importance.
Paclitaxel (PTX) is frequently used in all clinical chemotherapy protocols and is an important drug for the treatment of esophageal carcinoma; however, resistance has been observed in clinical practice. Resistance to PTX has been attributed to several mechanisms, including the overexpression of the P-gp efflux pump and resistance to apoptosis, among others (5,6). Consequently, these mechanisms force cells to dysregulate apoptosis, which facilitates tumor progression. The majority of chemotherapeutic drugs kill cancer cells via the induction of apoptosis; however, cancer cells can escape apoptosis or antagonize apoptosis. Some scientists believe that cells with the MDR characteristic often exhibit resistance to apoptosis, and resistance to apoptosis may be one of the essential characteristics of MDR (4,7,8).
Isodon rubescens, a Chinese herb, has been used as a folk botanical medicine in China for the treatment of cancer and inflammatory diseases for many years. Oridonin, an active diterpenoid compound found in Isodon rubescens, has been widely used in the treatment of human diseases ranging from inflammation to cancer (9,10). Jesridonin (JD; chemical structure shown in Fig. 1) is a diterpenoid compound that was obtained via the structural modification of oridonin. Recently, in a previous study, we demonstrated that JD exhibited strong antitumor activity in EC109 human esophageal carcinoma cells, both in vitro and in vivo, and demonstrated no adverse effects on major organs in nude mice (11). However, the anti-tumor activity of JD in paclitaxel-resistant cells remains unclear. To overcome the obstacles related to drug resistance and to improve the clinical outcomes of patients with esophageal squamous carcinoma (ESCC), in this study, we examined the effects of JD on the PTX-resistant EC109 human cancer cell line, EC109/Taxol, with the ultimate goal of identifying a novel therapy for paclitaxel-resistant ESCC.
Propidium iodide (PI), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bisbenzimide Hoechst 33258 were from Sigma (St. Louis, MO, USA). RNase A and the bicinchoninic acid (BCA) protein assay kit were from Solarbio (Beijing, China). The rabbit monoclonal antibodies against p53 (sc-393031), caspase-9 (sc-8355) and caspase-3 (sc-65497) are the products of Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit monoclonal antibodies against Bcl-2 (ZA-0536) and Bax (TA337090) were from Zhongshan Goldenbridge Biotechnology, Co., Ltd. (Beijing, China). Primary antibody against p53 upregulated modulator of apoptosis (PUMA; E1A5173) was from Nanjing EnoGene Biotechnology (Nanjing, China). Primary antibody against cleaved caspase-3 (#9661) and cleaved caspase-9 (#9501) was purchased from Cell Signaling Technology (Danvers, MA, USA). Peroxidase-conjugated affinipure goat anti-rabbit IgG (ZB-2301) and anti-mouse IgG (ZB-2305) IgG as secondary antibodies were from Zhongshan Goldenbridge Biotechnology, Co., Ltd.
The EC109 cell line was obtained from the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (both from HyClone, Beijing, China), 100 U/ml of penicillin and 100 µg/ml of streptomycin at 37°C in a humidified air atmosphere containing 5% CO2. The resistant cell line (EC109/Taxol) was developed in vitro by the intermittent exposure of the human ESCC cell line, EC109, to a high concentration of PTX, with a stepwise increase in concentration over a period of 6 months. These cells were developed, cultured and maintained as previously described (8).
JD was obtained from the New Drug Research and Development Center of Zhengzhou University. JD is a 7,14-acetal derivative of Oridonin (a natural antitumor compound isolated from Isodon rubescens). The chemical structure was confirmed using NMR, MS and IR data (Fig. 1). For the in vitro assay, JD was dissolved in DMSO and stored at −20°C. The concentration of DMSO in the culture medium was under 1% (v/v) and had no intrinsic effect on cell proliferation.
A total of 15 female 6-week-old BALB/c nu/nu mice weighing 18–19 g each were purchased from Hunan Slack King of Laboratory Animal, Co., Ltd. (Hunan, China) and maintained under specific pathogen-free (SPF) conditions at 25°C in an atmosphere with 50% humidity for the experiments. Lighting was operated automatically on a 12-h light/dark cycle. The mice were raised in a sterile environment and received adequate water and food. Throughout the trial period, all experiments strictly followed institutional guidelines and were approved by the Experimental Animal Care Committee of Zhengzhou University (approval no. SPS140302).
The cells (8×103 cells/well) were inoculated into each well in 96-well plates (Nest Biotechnology Co., Ltd., Wuxi, Jiangsu, China) in 100 µl of culture medium. Following an overnight incubation (37°C with 5% CO2), the medium was removed and replaced with various concentrations of JD. The plates were incubated at 37°C in 5% CO2 for 24, 48 and 72 h. Thereafter, 20 µl of MTT solution (5 mg/ml) were added to each well, and the plate was incubated for an additional 4 h at 37°C. The resulting formazan crystals were dissolved in 150 µl of DMSO following aspiration of the culture medium. The plates were read at 490 nm using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The 50% inhibitory concentration (IC50) represents the concentration of the modulators that was required for 50% inhibition.
The cells were seeded at 1.4×104 cells/well (in 24-well plates). Following an overnight incubation, the medium was removed and replaced by 10 µM of JD. Cells from 3 wells were trypsinized and separately counted at the time points of 24, 48, 72, 96, 120, 144 and 168 h following treatment with JD.
A colony formation assay was performed to determine the effects of JD treatment on the colony-forming ability of EC109/Taxol cells. Briefly, the cells were seeded into 6-well plates at 1×103 cells/well. Following an overnight incubation, the medium was removed and replaced by various concentrations of JD. Following incubation for 7 days, the cells were then fixed with 95% ethanol for 30 min and stained with 0.5% crystal violet for 30 min. Cell colonies (≥50 cells) were counted under an inverted light microscope (BDS15010071, Aote light microscope, Chongqing, China). The colony formation rate was calculated as follows: (colony counts)/(cells inoculated) ×100%.
The evaluation of JD activity was also carried out on nude mice bearing EC109/Taxol xenografts. The tumor model was established as previously described (8) by subcutaneously inoculating EC109/Taxol cells (1×107/mice) into the left armpits of the mice. The tumors were allowed to reach an average size of 100–150 mm3 (as estimated by Vernier caliper measurement), and the mice bearing the EC109/Taxol cells were then randomly divided into 3 groups. The 3 groups of mice were treated as follows: the mice in group C were the free control, the mice in group T1 were treated with 5 mg/kg of JD, and the mice in group T2 were treated with 10 mg/kg of JD (n=5 mice/group). The drug (the clathrate of JD was formed with β-cycloamylose and dissolved in 0.9% NaCl) and the vehicle (β-cycloamylose in 0.9% NaCl, 10 mg/kg) were administered by intravenous tail vein injection every 3 days for a total of 21 days. At the end of the treatment, the mice were sacrificed by cervical vertebrae dislocation, and the tumors were dissected and weighed. During this period, the body weights of the animals were recorded every 3 days, and the tumor sizes were measured by determining 2 perpendicular dimensions at the same time. The volume (V) of each tumor was then calculated using the following formula:
where length (a, mm) is the longest diameter and width (b, mm) is the shortest diameter perpendicular to the length (12,13). The calculated tumor volumes were expressed in percentages as relative tumor volume (RTV) using the following equation (14):
where Vn is the tumor volume at day n of treatment and V0 represents the initial tumor volume at the onset of treatment. The inhibition of cancer growth was defined as a ratio of the tumor weight compared to the vehicle control tumor weight. The specimens from the EC109/Taxol xenografts were removed for hematoxylin and eosin (H&E) staining.
The specimens of EC109/Taxol xenografts were fixed in 10% formalin and then processed by the usual technique of paraffin inclusion. The histological technique of paraffin inclusion used in this study was performed in the following sequence: dehydration, clarification, waxing and proper inclusion. The embedded tissues were cut into 4-µm-thick sections, affixed to glass slides, deparaffinized in xylene, rehydrated in ethanol, rinsed in distilled water and then fixed with 4% formaldehyde. Thereafter, the paraffin sections were stained with H&E, dehydrated in graded alcohol, cleared in xylene, cemented with neutral resin. General morphologies were viewed under a light microscope (347655; Leica Microsystems CMS GmbH, Wetzlar, Germany).
The cells were seeded in a 6-well plate and treated with JD at the indicated concentrations for 24 h. The cells were then washed with phosphate-buffered saline (PBS) 3 times followed by fixation with 4% paraformaldehyde for 15 min, then washed 3 times with PBS. Hoechst 33258 was subsequently added at a final concentration of 10 µg/ml in the dark. Thirty minutes later, the cells were carefully washed twice with PBS. The apoptotic cells were examined and identified according to the condensation and fragmentation of their nuclei under a fluorescence microscope (Eclipse TE2000-S; Nikon, Tokyo, Japan).
To determine the effect of JD on the cell cycle, 5.0×106 EC109/Taxol cells in the exponential growth phase were treated with various concentrations of JD for 24 h. After an incubation period, the cells were collected, centrifuged and fixed with ice-cold 70% ethanol at 4°C overnight. The cells were washed with PBS before staining with PI and then suspended in staining buffer (50 µg/ml PI, 1% Triton X-100 and 0.5 mg/ml RNase A in PBS) at 37°C for 30 min in the dark. The cells were analyzed on an Accuri C6 flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). The histograms of DNA distribution were shown as a sum of cell populations at the G0/G1, G2/M or S phase using FlowJo software.
An Annexin V-fluorescein isothiocyanate kit (Annexin V- FITC; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was used to stain and identify the percentage of apoptotic cells. Briefly, the cells were seeded into 6-well plates and treated with 0, 10, 20 and 40 µM of JD for 24 h. The cells were then harvested, washed with ice-cold PBS and resuspended in binding buffer containing Annexin V-FITC (0.5 mg/ml) and PI (0.5 mg/ml). Following 15 min of incubation at room temperature in the dark, the stained cells were analyzed immediately by an Accuri C6 flow cytometer (Becton-Dickinson). Apoptotic cells were determined by cells that were both Annexin V-positive and PI-negative.
Western blot analyses were conducted as described previously (8). The blots were then probed with each of the following antibodies: anti-p53 antibody (1:1,000), anti-Bcl-2 antibody (1:500), anti-Bax antibody (1:1,000), anti-PUMA antibody (1:500), anti-procaspase-3 antibody (1:1,000), anti-cleaved-caspase-3 antibody (1:250) and anti-procaspase-9 antibody (1:1,000). After washing the membrane with PBST (PBS, 0.05% Tween-20) 3 times (10 min each), the blots were subsequently incubated with a goat anti-mouse or anti-rabbit IgG-HRP secondary antibody (1:10,000) at 37°C for 2 h. The antibody-reactive bands were revealed by an enhanced chemiluminescence substrate and were exposed to Kodak radiographic film.
All statistical analyses were carried out using the SPSS 17.0 statistical packages. The data are expressed as the means ± standard deviation (SD). Continuous variables were analyzed using the Student's t-test. A value of P<0.05 was considered to indicate a statistically significant difference, and a value of P<0.01 a highly statistically significant difference.
Our initial experiment was carried out to determine the inhibitory effect of JD on EC109 and EC109/Taxol cells by MTT assay. The results revealed that JD displayed similar IC50 values against drug-sensitive EC109 cells and paclitaxel-resistant EC109/Taxol cells in vitro and indicated that JD had a potent growth inhibitory effect on both of these cell lines in a concentration- and time-dependent manner (Fig. 2A and B and Table I).
The activity of JD was further evaluated using the colony formation assay. As shown in Fig. 2C and Table II, JD not only significantly inhibited the proliferation of EC109/Taxol cells, but also reduced the colony-forming ability compared with the control groups. The colony formation assays demonstrated that JD treatment at concentrations as low as 0.5 µM significantly suppressed the colony-forming activity of EC109/Taxol cells. According to the inhibitory effects of JD on the colony-formaing ability of EC109/Taxol cells (Table II), we calculated that the IC50 value of JD was 0.87 µM using SPSS software. The data also indicated that the JD-induced suppression of colony formation correlated well with the concentrations of JD, suggesting that JD treatment inhibited the colony-forming ability of EC109/Taxol cells in a dose-dependent manner.
In addition, proliferation curves were generated for the EC109/Taxol and EC109 cells incubated with 10 µM of JD. The results indicated that 10 µM JD induced significantly decreased the proliferation of EC109/Taxol and EC109 cells. After 48 h and 72 h of incubation, the numbers (×104) of the EC109/Taxol (48 h-0.219±0.03, 72 h-0.125±0.01) and EC109 (48 h-0.563±0.02, 72 h-0.025±0.01) cells were only a few. Following 96 h of incubation, the proliferation of the EC109/Taxol and EC109 cells was completely inhibited (Fig. 2D).
To evaluate the in vivo protective effects of JD, we used the growth of EC109/Taxol cell xenografts in female nude mice as an in vivo model. Five animals per treatment group, injected intravenously, were used. No significant difference was observed in the body weight changes among the different treatment groups, suggesting that this regimen was safe (Fig. 3A). Compared with the control group, the group treated with JD (either 5 or 10 mg/kg) demonstrated a significantly inhibition of tumor growth, both in terms of tumor size and weight (Fig. 3B and C).
Histological analysis of the H&E-stained tumor sections from the EC109/Taxol xenografts from the mice treated with JD demonstrated a marked change in tissue and cell morphology compared with those from the vehicle control group (Fig. 3D). These changes included a low cell density and multinucleated cells with condensed chromatin staining and pyknosis, indicating mitotic catastrophe and apoptosis.
The cellular DNA content of JD-treated cells and untreated cells was analyzed by flow cytometry to detect changes in the cell cycle distribution of the EC109/Taxol cells. As shown in Fig. 4 and Table III, treatment of the EC109/Taxol cells with JD at 10, 20 and 40 µM for 24 h significantly increased the G2/M cell population up to 33.95±1.97, 48.29±1.38 and 69.169.10±4.02%, respectively, whereas 25.56±2.02% of the control cell population was in the G2/M phase. The increase in the G2/M cell population induced by JD occurred at the expense of a decrease in the G0/G1 and S cell populations. These results indicated that the cells treated with JD exhibited a G2/M phase arrest.
Following treatment with JD at the indicated concentrations for 24 h, morphological changes in EC109/Taxol cells were assessed using an inverted microscope. DMSO (0.1%) was used as a control. The EC109/Taxol cells treated with JD exhibited apoptosis-related morphologies, such as cell shrinkage and rounding up, cell membrane blebbing and nuclear fragmentation and condensation (Fig. 5A). In addition, nuclear morphological changes at the corresponding concentration were also recorded and qualitatively evaluated by means of Hoechst 33258 fluorescent staining following incubation for 24 h. Typical apoptotic markers, including nuclear fragmentation and nuclear condensation, were detected, particularly at the highest concentrations of JD (Fig. 5B).
In addition, to determine whether JD treatment induces apoptosis, cells incubated with increasing concentrations of JD for 24 h were analyzed using flow cytometry to assess the diploid DNA content and for phosphatidylserine (PS) exposure to the cell surface. The percentages of live, early-apoptotic, late-apoptotic and necrotic cells were calculated using an Accuri C6 flow cytometer. Treatment of the EC109/Taxol cells for 24 h with 10, 20 and 40 µM of JD resulted in a dose-dependent increase in the percentage of the apoptotic population up to 6.7±1.80, 36.2±3.60 and 46.1±2.62%, respectively, compared with the control treated cells (3.6±0.64%) (Fig. 5C and D).
As p53 is a major tumor suppressor, we examined the role of p53 in JD-mediated growth inhibition. JD treatment resulted in an increase in the p53 protein levels in the EC109/Taxol cells (Fig. 6A and B). Apoptosis is a well-organized cellular event, characterized by the activation of caspase cascades. Caspases are synthesized as pro-enzymes that must be activated to perform their functions. Caspase-9 is the major caspase member that regulates the intrinsic apoptosis pathway, whereas caspase-3 is one of the critical effector caspases in the downstream execution of apoptosis (5,15). As shown in Fig. 6A, F and G, upon treatment with JD, the procaspase-9 and procaspase-3 levels decreased, and the cleaved-caspase-9 and cleaved caspase-3 levels increased, indicating the activation of caspase-9 and caspase-3.
We also examined the expressions levels of Bax and Bcl-2 and then analyzed the ratio of Bax/Bcl-2. The results of western blot analysis revealed that Bcl-2 expression was significantly decreased and that Bax expression was not altered in the JD-treated EC109/Taxol cells (Fig. 6A and C). Statistical analysis also indicated that JD in the range of 10-40 µM increased the ratio of Bax/Bcl-2 (Fig. 6D). Moreover, PUMA expression was upregulated following treatment with JD (Fig. 6A and E).
The EC109/Taxol cells, a paclitaxel-resistant cell line expressing high levels of P-gp, were generated from the drug-sensitive parental EC109 cells by the intermittent exposure to a high concentration of PTX in stepwise time increments over a period of 6 months. The PTX resistance of the EC109/Taxol cell line was approximately 67.2-fold that of the EC109 cell line. In addition, the EC109/Taxol cells exhibited a greater cross-resistance to 5-fluorouracil (5-FU), cisplatin (CDDP) and epirubicin (EPI) than its parental EC109 cell line (8). In this study, we examined the effect of JD on EC109/Taxol cells both in vitro and in vivo. JD exhibited a strong activity against EC109/Taxol cells. The JD-induced inhibition of cancer growth was confirmed in nude mouse models. The results indicated that EC109/Taxol xenografts were sensitive to JD.
Cell cycle perturbation plays an important role in carcinogenesis, and cell proliferation, apoptosis, differentiation and senescence are all cell cycle-dependent (16). In the next set of experiments, we examined the effects of JD on the cell cycle distribution of EC109/Taxol cells. EC109/Taxol cells were treated with JD at various concentrations (0, 10, 20 and 40 µM) for 24 h. JD caused an accumulation of cells in the G2/M phase and diminished cells in the S and G0/G1 phases compared with the untreated control cells. The results suggested that JD caused an obvious G2/M arrest pattern in a concentration-dependent manner, with a concomitant decrease in terms of the number of cells in other phases of the cell cycle.
Apoptosis is a regulated type of physiological cell death in which a controlled sequence of events leads to cell elimination essential for the maintenance of homeostasis (17,18). As the death of tumor cells by chemotherapy is mediated mainly by the induction of apoptosis, the deregulation of apoptosis would confer resistance to anticancer agents. In this study, apoptosis, as determined using Hoechst 33258 staining and flow cytometric analysis (Fig. 5), was confirmed to be responsible for the JD-mediated anti-resistance activity in EC109/Taxol cells. Thereby, our research provides new approaches to overcoming drug resistance by inducing apoptosis in resistant EC109/Taxol cell lines.
For further study, we elucidated the possible mechanisms responsible for the apoptosis induced by JD. Although the mechanism of drug resistance is not completely defined, the deficiency in apoptosis and abnormally activated survival signaling pathways have been observed in most cancers with MDR. Apoptosis deficiency may be involved in cancer initiation, progression and treatment failure in cancers with MDR (19). Apoptosis is regulated at multiple levels in the extrinsic and intrinsic pathways (20). The extrinsic pathway involves death receptors on the cell surface that can directly activate caspase-8. The intrinsic pathway of apoptosis centers on the mitochondria and is associated with the loss of the mitochondrial membrane potential (MMP), the release of cytochrome c into the cytosol and the activation of caspase-9/3 (21,22). The Bcl-2 and caspase families are considered as the most important proteins for regulating apoptosis in the intrinsic pathway. The Bcl-2 family can be divided into two types: anti-apoptotic and pro-apoptotic proteins (23). The ratio of Bcl-2/Bax is a critical determinant for cell apoptosis. The dysregulation of the Bcl-2 family has been shown to induce the destruction of the mitochondrial membrane, which is accompanied by the release of intramembranous proteins into the cytosol, such as cytochrome c and other apoptosis-inducing factors, and to subsequently induce the activation of the caspase cascade by activating caspase-9 and caspase-3 (24,25). Bax is constitutively located within the cytosol, whereas the activation of Bax involves its translocation from the cytosol to the outer mitochondrial membrane and, subsequently, its insertion into the membrane (26). Bax can release cytochrome c after the formation of the Bax/Bak hetero-oligomer, whereas Bcl-2 can interact with activator proteins or Bax/Bak, thereby sequestering these proteins (27-29). The BH3-only protein PUMA is a pro-apoptotic protein that is normally expressed at a low level. Its upregulation leads to cytochrome c release, which results in apoptosis (30). The tumor suppressor and transcription factor p53 is a key modulator of cellular stress responses, and the activation of p53 can trigger apoptosis in many cell types (31,32). Therefore, the dysregulation of the Bcl-2 and caspase families and of p53, as well as their respective signaling pathways, has been considered the most important mechanism in the development of apoptotic deficiency and the phenotype of MDR in cancer cells. The regulation of these protein kinases may be an effective regimen for the treatment of cancers with apoptotic deficiency and cells with MDR (33).
Our results indicated that treatment with JD significantly downregulated anti-apoptotic Bcl-2 protein expression, upregulated PUMA expression and did not alter pro-apoptotic Bax protein expression, thus shifting the Bax/Bcl-2 ratio in favor of apoptosis. Moreover, JD upregulated the expression of p53, cleaved-caspase-9 and cleaved-caspase-3 in EC109/Taxol cells and downregulated the expression of procaspase-3 and procas-pase-9 in EC109/Taxol cells in a concentration-dependent manner. We therefore suggested that the induction of apoptosis in EC109/Taxol cells by JD may be due to the activation of the mitochondria-mediated intrinsic apoptosis pathway. Further research on the specific mechanisms of action and the efficacy of JD in other cancer cell lines is currently underway.
In conclusion, to the best of our knowledge, our results demonstrate for the first time that JD exerts an anti-proliferative and anti-MDR effect on EC109/Taxol cells. JD treatment via intravenous injection in mice effectively prevented the growth of EC109/Taxol xenografts without exerting any significant toxicity. These findings suggest the possibility that JD may be a promising drug candidate and provide new supporting evidence for the manipulation of the dysregulated apoptotic pathway as a strategy with which to overcome drug resistance.
This study was supported by the National Natural Science Foundation of China (project nos. 21372206 and 81430085, for H.L.), the Natural Science Foundation of Henan Province of China (the foundation and frontier technology research program) (project no. 152300410030 for C.W.), the Science and Technology Research Key Project in the Henan province Department of Education (project no. 14B350011 for C.W.) and the Specialized Research Foundation of the Doctoral Program of High College for new teachers (no. 20104101120012 for C.W.).