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Zuo Jin Wan reverses P-gp-mediated drug-resistance by inhibiting activation of the PI3K/Akt/NF-κB pathway
- Hua Sui†1,
- Shu-Fang Pan†2,
- Yu Feng3,
- Bao-Hui Jin1,
- Xuan Liu1,
- Li-Hong Zhou1,
- Feng-Gang Hou4,
- Wen-Hai Wang1,
- Xiao-Ling Fu1,
- Zhi-Fen Han1,
- Jian-Lin Ren4,
- Xiao-Lan Shi2,
- Hui-Rong Zhu1 and
- Qi Li1Email author
© Sui et al.; licensee BioMed Central Ltd. 2014
Received: 11 January 2014
Accepted: 14 July 2014
Published: 1 August 2014
Zuo-Jin-Wan (ZJW), a traditional Chinese medicine formula, has been identified to be effective against drug resistance in cancer. In the present study, we investigated the effect of ZJW on acquired oxaliplatin-resistant and the PI3K/Akt/NF-κB pathway in vitro.
We tested the dose–response relationship of ZJW on reversing drug-resistance by CCK-8 assay and flow cytometry analysis in vitro. The protein expression of P-gp, MRP-2, LRP, and ABCB1 mRNA expression level were evaluated by Western blot and quantitative RT-PCR. The activities of PI3K/Akt/NF-κB pathway were also examined with or without ZJW, including Akt, IκB, p65 and their phosphorylation expression.
We found that ZJW significantly enhanced the sensitivity of chemotherapeutic drugs and increased oxaliplatin (L-OHP)-induced cell apoptosis in a time- and dose-dependent manner. Moreover, both ZJW and a PI3K specific inhibitor (LY294002) suppressed phosphorylation of Akt (Ser473) and NF-κB, which is necessary in the activation of the PI3K/Akt/NF-κB pathway. The effect of ZJW in reversing drug-resistance and suppressing phosphorylation of Akt (Ser473) and NF-κB were weakened after treatment with a PI3K/Akt activator in HCT116/L-OHP cells.
Our study has provided the first direct evidence that ZJW reverses drug-resistance in human colorectal cancer by blocking the PI3K/Akt/NF-κB signaling pathway, and could be considered as a useful drug for cancer therapy.
Colorectal cancer (CRC) is the second commonest cause of cancer-related deaths in the Western World, and resistance to chemotherapy remains the primary reason for treatment failure in advanced CRC . Chemotherapy is an important therapeutic method for CRC patients. However, the development of drug resistance usually results in the failure of chemotherapy . Drug-resistance of a tumor can be divided into targeting resistance, which arises to one or two special drugs, and multidrug resistance (MDR), which means the resistance of cancer cells to one chemotherapeutic drug accompanied by resistance to other chemotherapeutic drugs that may have different structures and mechanisms of action .
To date, the most important potential mechanism of drug-resistance is the ABC transporter family, including the well known P-glycoprotein (P-gp, encoded by the ABCB1 gene), MDR-associated protein 1 (MRP1, encoded by the ABCC1 gene) and ABC subfamily G member 2 . To reverse cellular transport protein-mediated drug-resistance, studies have been conducted to explore possible mechanisms via signaling pathways, including phosphatidylinositol 3-kinase (PI3K) signal transduction.
The phosphoinositide 3-kinases (PI3Ks), a family of lipid kinases that propagate intracellular signaling cascades regulating a wide range of cellular processes, are believed to be one of the reasons for the development of chemoresistance during cancer therapy . PIP3, which is a PI3K phosphorylation product, brings two PH domain-containing serine/threonine kinases, phosphoinositide-dependent kinase 1 (PDK1), and AKT into close proximity . Previous studies have also indicated that Akt phosphorylation can induce drug-resistance in several kinds of tumors, such as ovarian cancer , breast cancer , and hepatocellular carcinomas . Furthermore, AKT impedes negative regulation of the transcription factor NF-κB, leading to increased transcription of antiapoptotic and prosurvival genes . In fact, spontaneous activation of NF-κB has been detected in human colorectal cancer tissues, and activation of NF-κB is believed to result in the chemoresistant phenotype in colorectal cancer cells [4, 11].
Zou-Jin-Wan (ZJW), a traditional Chinese medicine formula, has been used as a therapy to slow colorectal cancer progression, improve quality of life, and prolong survival times. ZJW is composed of Rhizoma Coptidis and Fructus Evodiae in the ratio 6:1 (w/w). Coptis chinensis Franch, the main component of Rhizoma Coptidis, has been reported to reverse drug-resistance cancer [12, 13] and suppress the nuclear translocation of p50/p65 NF-κB proteins and their binding to target genes . Fructus Evodiae and its major alkaloid component, evodiamine, have been demonstrated to induce apoptosis in human melanoma A375-S2 cells, lead to inactivation of the PI3K/Akt/NF-κB pathway, and stop the translocation of NF-κB . Although the role of Rhizoma Coptidis and Fructus Evodiae in the PI3K/Akt/NF-κB pathway have been investigated extensively, much less is known about their potential role in regulating drug-resistance when mixed in ZJW.
Although the ZJW herbal formula has anti-cancer effects, its underlying mechanism in reversing drug-resistance remains unknown. In this study, we aimed to elucidate the effect and the molecular mechanisms of the Chinese herbal formula, ZJW, in human cancer cells in vitro.
Preparation of the extracts for ZJW
Rhizoma Coptidis and Evodia were purchased from Lei-Yun-Shang Pharmaceutical Group (Shanghai) and identified as the coptis of ranunculaceae and the fruit of Tetradium, respectively. They were formally identified by Professor DZ Wu (The School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, China).
ZJW was formulated by Rhizoma Coptidis and Evodia (in a ratio of 6:1). All the herbs were purchased from ShuGuang Hospital herb store and were done as described previously . Briefly, the mixture (70 g) was extracted twice for 1 h each time by refluxing in ethanol (1:8, v/v). The filtrates were concentrated and dried in vacuum at 60°C. The concentrated extract was then dried by lyophilization to obtain the ZJW extract at a yield of dried powder of 24.4%. The extract was stored at 4°C, and its preparations were standardized, regulated and quality controlled according to the guidelines defined by Chinese State Food and Drug Administration (SFDA).
Cell culture and reagents
Human colorectal cancer HCT116 cells were purchased from the Shanghai Cell Collection (Shanghai, China). The HCT116/L-OHP MDR cell lines were established and maintained in our laboratory. Cells were grown in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA) at 37°C in a 5% CO2 humidified atmosphere. HCT116/L-OHP cells were routinely maintained in a medium containing 5,000 ng/mL oxaliplatin (L-OHP). Monoclonal antibodies against P-gp, MRP-2, LRP, IκB, Akt, p65, and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA).
Cell viability assays
Cell proliferation was determined using the cell counting kit, CCK-8. Briefly, cells were seeded in 96-well plates at 1 × 104 cells/well. When the cells reached 60% confluence, the medium was removed and replaced with fresh medium containing varying concentrations of ZJW or its mixture with anti-tumor drug (L-OHP) and incubated for 48 h. The CCK-8 assay was then performed: after 2 h of incubation with culture medium containing the CCK-8 reagent, the absorbance was read at 450 nm using a microplate assay reader (Labsystems Dragon, Wellscan). Relative inhibitory rate of cell growth was calculated according to the formula: R = (A2 − A1)/A2 × 100% and P = A1/A2 × 100%, in which R was relative inhibitory rate, P was relative proliferation ratio of cell growth, A1 is the mean absorbance of transfected cells, and A2 is the mean absorbance value of untransfected control cells without drug treatment. All experiments were done with five wells per experiment in triplicate.
Apoptosis and cell-cycle assay
Cells were seeded in 6-well plates (4 × 105 cells/well). After 12 h, three dose concentrations of ZJW were added. Flow cytometry was used to detect apoptosis by determining the relative amount of AnnexinV-FITC-positive-PI-negative cells, as previously described . Unstained cells, cells stained with Annexin V-FITC alone, and cells stained with propidium iodide alone were used as controls. Singly stained cells were used to adjust electronic compensation on the FL1 and FL2 channels. After apoptosis assay, cell cycle distributions were analyzed with the ModFit program (BD, San Diego, CA, USA). All samples were assayed in triplicate, and the fraction of each cell cycle phase was calculated.
Western blot analysis
Whole cell lysate for SDS-PAGE and western blot analysis for P-gp, MRP-2, LRP, IκB, Akt, p65, and phosphorylation of Akt and IκB expression was prepared as previously reported . The lysate was incubated on ice in immunoprecipitation assay buffer for 2 h before being homogenized using a mortar and pestle. The homogenized sample was centrifuged, and the supernatant was collected and stored at −80°C. Equal loading was confirmed with GAPDH. Densitometric analysis was done using the Scion Imaging software (Scion Corporation), with GAPDH as internal control.
Quantitative RT-PCR analysis
Tumor cells were homogenized and suspended with an RNAspin Mini Kit (GE Healthcare, Waukesha, WI, USA) for RNA isolation according to the manufacturer’s instruction. For cDNA synthesis, 1 μg of total RNA was reverse-transcribed using oligo-dT primers and the Superscript Amplification System (Life Technologies, Carlsbad, CA, USA). Quantitative RT-PCR was carried out using SYBR Green PCR Master Mix (Life Technologies). The PCR parameters consisted of initial polymerase activation at 94°C for 5 min, followed as previously described . Amplification of GAPDH RNA, a relatively invariant internal reference was performed in parallel, and cDNA amounts were normalized to equivalent GAPDH mRNA levels. Oligonucleotide primers for ABCB1 and GAPDH were as follows: Oligonucleotide sequence of ABCB1 (379 bp), F: 5′-TAATGCGACAGGAGATAGG-3′, R: 5′-TGCCATTGACTGAAAGAAC-3′, Oligonucleotide sequence of GAPDH (306 bp), F: 5′-ACCCACTCCTCCACCTTTGA-3′, R: 5′-CTGTTGCTGTAGCCAAATTCGT-3′.
ABCB1 promoter activity by vector transient transfection and dual luciferase assay
Transfection procedures were performed according to manufacturers’ instructions, with Lipofectamin 2000 as transfection reagent (Invitrogen). Briefly, 2 × 103 cells were plated in each well of a 96-well plate and incubated overnight. A mixture of Lipofectamine 2000 (10 nM) with ABCB1 promoter recombinant vector pGL3-Basic-ABCB1 promoter (0.8 μg/well) was added, followed by a 48 h incubation in regular medium. Then cells were harvested and analyzed using a dual-luciferase assay kit (Shanghai Lai’an Biotech. Co., Ltd, Shanghai, China) as previously reported .
Chromatin immunoprecipitation (ChIP) was done as described previously . N-ethylmaleimide was added to the cell lysis buffer at a final concentration of 10 mM to preserve poly-ubiquitinated protein conjugates.
All experimental data are expressed as mean ± standard deviation of at least three independent experiments performed in duplicate. Significance was determined by a one-way analysis of variance (ANOVA) and Holm’s multiple-comparison test. Statistical significance was set at a P-value of less than 0.05. All analyses were carried out using SPSS13.0 (SPSS, Chicago, IL, USA).
ZJW modulates L-OHP resistance in vitro
The effect of ZJW and PI3K/Akt pathway on L-OHP resistance
ZJW inhibits P-gp expression and the effect of the PI3K/Akt pathway
ZJW suppresses P-gp mediated drug-resistance by inhibiting activation of the PI3K/Akt/NF-κB pathway in vitro
The aim of this study was to investigate whether the anti-drug-resistance effect of ZJW was via inhibition of PI3K/Akt/NF-κB signaling in colorectal drug-resistance cancer. Our previous studies demonstrated that ZJW could reverse the MDR phenotype by increasing the sensitivity of MDR cells to chemotherapeutic agents and inhibiting P-gp expression both in vitro and in vivo. In the present study, we investigated the anti-cancer molecular mechanisms of ZJW ethanol extracts in signal pathways reversing drug-resistance.
In colorectal cancer, patients who do not respond to chemotherapy usually have a high expression of various ABC transporter pumps, which are located on the cytoplasmic side of the resistant cell membrane, resulting in an increased drug efflux . In addition, the effectiveness of current chemotherapeutic agents is limited by drug resistance, thus the discovery of the mechanisms governing the cellular response to chemotherapy and develops a new strategy for treatments are priority . Traditional Chinese prescriptions and formulae can be used as a therapy to effectively control cancer progression, improve quality of life, and prolong survival times instead of conventional chemotherapy [19–21].
Several studies have demonstrated that the PI3K/AKT pathway activates the NF-kB system, which could lead to increase the transcription of target genes such as ABCB1 , COX-2 , and p53 . Therefore, a definitive relationship between the PI3K/AKT/NF-kB pathway and drug-resistance has yet to be established . However, no report has examined the effect any drug on the reversal of drug-resistance via blockage of the PI3K/Akt/NF-kB signal pathway in colorectal cancer.
ZJW, a traditional Chinese herbal medicine, is viable and effective for inhibiting drug-resistance in lowest dosage of the IC10 to MDR cells . In the present study, the concentration range of 50 μg/mL (IC10 to ZJW) was also used to help enhance the sensitivity of chemotherapeutic agents in the cell proliferation, apoptotic, but not in cell cycle. However, the mechanism of ZJW in the resistance to MDR cancer remains unknown. Earlier reports showed that evodiamine, a component of ZJW, could inhibit the PI3K/Akt pathway and targeted NF-κB in pancreatic cancer . In our study, a remarkable activation of phosphorylation AKT and NF-κB was detected in HCT116/L-OHP cells, which also have up-regulation of P-gp. In addition, we showed that the levels of phosphorylation AKT and NF-κB in HCT116/L-OHP cells decreased after exposure to ZJW. However, the effects of ZJW in inhibiting of the level of P-gp, phosphorylation AKT, and NF-κB were weakened after treatment with the PI3K/Akt activator in HCT116/L-OHP cells. Therefore it is reasonable to believe that ZJW might play a suppressive role in the expression of P-gp via inhibition of the PI3K/Akt/NF-κB pathway.
These results are consistent with previous reports and may explain the effects of ZJW, including enhancing the inhibition rate of chemotherapeutic agents in a time-dependent manner, and helping chemotherapeutic drugs induce cell apoptosis in a dose-dependent manner. These results also agree with in vivo data that showed that ZJW was able to reduce tumor volume and induce cell apoptosis with the combination of chemotherapeutic agents . Therefore, ZJW exhibits down-regulation of the expression of ABCB1/P-gp via inhibition of the PI3K/Akt/NF-κB pathway in vitro.
This work was supported by the National Natural Science Foundation of China (No. 81202812, 81373862 and 81403360), Program of Shanghai Municipal Education Commission (No. 09YZ132, 2011JW57, and 12YZ058), and Shanghai Municipal Health Bureau (No. 2011ZJ030, 20114Y013, 2010QL050B, and 20114Y001).
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