Honokiol Affecting Cell Proliferation, Apoptosis And Reversing Drug Resistance Of Leukemic Cell Line U937 Cells

Background: Apoptosis or programmed cell death (PCD) is a process of active cell death controlled by genes, by which the organism maintains self-stabilization and regulates the equilibrium between proliferation and death of body cells. Apoptosis appeared with specific morphologic and biochemical features but with no evident inflammation, which are the straight results of the specific substrates-decompounding induced by caspases. Bcl-2 family members are the key regulators of apoptosis, which consists of two sub-families:pro-survival members (such as Bcl-2, Bcl-XL) and pro-apoptosis members (such as Bax, Bid, Bak). The ratio of these two sub-families'activity determines cells'fate. Increased Bax/Bcl-2 ratio will trigger downstream caspase and inducing apoptosis. Escaping from apoptosis is an important characteristic of leukemia cells. Inducing apoptosis of leukemic cells has become an important strategy in leukemia therapy.New chemotherapeutic agents and hemopoietic stem cell transplant have significantly enhanced the short and long term curative effects and improved the prognosis of leukemia patients, but some patients developed primary or secondary multidrug resistance (MDR) which results in chemotherapy failure. MDR is one of the most important reasons for the failure of leukemia chemotherapy. MDR has a special broad-spectrum drug resistant characteristic. Once cancer cells develop resistance to a certain anticancer drug, they will also have resistance to many other drugs which even have unrelated structure and unrelated functional mechanism.The mechanisms of MDR are extremely complicate with multiple factors involved in. The definitive mechanisms are shown as following: 1. The over- expression of membrane protein acting as drug pump, such as multi drug resistance protein1 (mdr1), multi-drug resistance related protein (MRP), nucleoprotein Lung resistance-related protein(LRP),Brest cancer resistance protein (BCRP). These proteins pump drugs out of cells and reduce the intercellular drug concentration. 2. The abnormal expression of enzymes, such as topoisomerase II (Topo II), Glutathione transferase-π(GST-π) and protein kinase C, etc. These enzymes participate in the action of multiple anti-cancer agents, so the change of their expressions must influence the therapeutic effect.3. The over-expression of anti-apoptosis genes, such as bcl-2 and C-myc, etc, cause cells to not be sensitive to apoptosis signals induced by drugs.In recent years, it has always been a hopeful strategy in the treatment of tumors by inducing apoptosis of tumor cells and reversing multidrug resistance with Chinese traditional medicine. Therefore, studis on the therapy of tumors by Chinese traditional medicine would be a hot topic in the field of oncology in the following years. Honokiol (HNK), a hydroxylated biphenyl compound isolated form the stem bark of Chinese herb Magnolia officnalis (Magnoliaceae), has been used to treat acute pain, cough, anxiety and gastrointestinal disorders in Eastern Asia. Various pharmacological effects of HNK are reported including an anti-inflammatory effect, an anti-microbial effect, inhibition of central neurons and scavenging of hydroxyl free radicals. In recent years, in vivo and vitro anti-cancer or apoptosis-inducing effects of honokiol on some solid tumors have been studied. But the proliferation- inhibiting or apoptosis-inducing effects of honokiol in vivo and in vitro have not been investigated yet. In addition, some studies showed that honokiol also has the ability of reversing drug resistance in Multiple Myeloma cells. However, there is not report about whether honokiol has the ability of reversing multidrug resistance in leukemia cells with multidrug resistance.Establishing a multidrug resistance cell line is essential for investigating the mechanism of MDR and finding out the methods to reverse MDR. In previous studies, most of multidrug resistant cell lines were established by successive exposure a certain cell line to low dose of an anti-tumor drug, and increasing the dosage of this drug gradually, which is different from the common protocol of clinical chemotherapies, which use high dose drugs periodically.U937 cell line was established in 1976, whose genetic background was distincted and biological character was stable. Adriamycin (ADR) is one of the mostly used anti-tumor drugs. The functional mechanism of ADR is to stabilize the cleavable complexes of DNA and to inhibit the repairing of DNA, which led to DNA damage and cell death.Therefore, in the present study, U937 leukemic cells were exposed to high dose of ADR periodically and an ADR-resistant cell line was established and named U937/ADR.In order to explore the anti-leukemia effect of honokiol and its mechanism in vitro, we systematically observed the growth inhibition and apoptotic process of U937 leukemia cells treated with honokiol, detected the expression of apoptosis- and drug resistance- related genes and proteins, such as Bcl2, Bax, P-gp, MRP1,LRP,BCRP,GST-πand NF-κB in U937 cells and in U937/ADR cells.To date, there is no report on establishing U937-MDR cell line induced by ADR and the reversing multidrug resistance effect of honokiol in U937/ADR cells. So the establishment of U937/ADR provided not only a good model to investigate the mechanism of leukemic MDR in cellular and molecular levels, but also an essential subject to study how to reverse MDR. The anti-leukemia effect and multidrug resistance reversing effect of honokiol demonstrated in the present study showed that Honokiol may be an effective anti-leukemia drug.Methods1 The growth inhibition effect of honokiol on U937 leukemic cells in vitro.1.1 Culture U937 leukemic cell U937 Cells were cultured in RPMI-1640 with 10% new born bovine serum medium, 100 IU/ml penicillin and 100μg/ml streptomycin, and in standard conditions (37℃, 5% CO2, fully humidified atmosphere).1.2 Using Methyl thiazolyl tetrazolium (MTT) method to measure the growth inhibition rate of U937 and PBMCs treated with HNKU937 leukemic cell and PMNCs (Peripheral Blood Mononuclear Cells) from healthy volunteers were seeded in 96-well plate, treated with different concentrations of honokiol for 24 and 48 hours, then MTT added. The cells were cultured for 4 hours, the supernatant medium discarded. After the addition of DMSO, the absorbance value (A value) of each well was measured at 490 nm. Based on the formula: inhibition ratio=(control A value-experiment A value) / experiment A value×100%, the growth inhibition rate was calculated.1.3 Detecting cell cycle distribution of U937 cells treated with honokiolHonokiol treated U937 cells were fixed with 70% alcohol, stained with 1ml (50μg/ml) propidium iodide for 30 min, filtered with 35 mm nylon mesh, the DNA content and cell cycle distribution was analyzed by flow cytometry.1.4 Observing the morphology changes of U937 cells treated with honokiol, under transmission electron microscopeU937 leukemic cell was treated with different concentrations of honokiol for 10 hours. After the treatment, cells were collected, washed with PBS, and fixed with 2% glutaraldehyde, washed with PBS, fixed with 1% osmic acid, dehydrated with a series of acetone, embedded in epoxide resin, cut into ultrathin section, and stained with 2% acetic acid oil and lead citrate. The morphology changes of these cells were observed under transmission electron microscope—HITACHI 1200ES.1.5 Detecting the apoptosis of U937 treated with honokiol, by FCM with Annexin V/PI double stainingThe cultured U937 leukemic cell and normal PBMCs treated with various concentrations of honokiol for 24 and 48 hours, and then the cells were collected, suspected in binding buffer, added AnnexinV, left at room temperature for 15minutes. Finally, binding buffer and propidium iodide (PI) were added just before detecting the apoptosis by FCM .1.6 Measuring the activity of caspase-3U937 cells were treated with different concentrations of HNK for different time, then the cells were collected and suspended with cool cell lysis buffer. After the protein extracted, the protein concentration was detected, and the DEVD-pNA was added to the protein, warmed for 1h. Finally the A value of OD405 was measured, and the activity of caspase-3 calculated.1.7 Monitoring the effect of mitochondrial permeability transition pore inhibitor on cell growth.U937cells at the density of 1×106 was cultured with HNK or with the inhibitor of mitochondrial permeability transition pore, Cyclosporin A, at a final concentration of 5μM for 2h, then 10μg/ml HNK was added to the culture and the cells were cultured for another 6h or 12h. At the end point of culturing, PI was added to the cells and the growth inhibition rate of U937 cells, treated with Cyclosporin A, HNK or Cyclosporin A together with HNK, was measured by MTT.1.8 Detecting the expression level of bcl-2 and bax mRNA in U937 cells after the treatment with honokiol by RT-PCR.Total RNA was isolated from HNK treated U937 cells by Trizol. The reverse transcription reaction was performed with M-MLV and Random primer. The specific primers were used to amplify the target gene. The products were analyzed with agarose gel electrophoresis and stained with goldview, the integral optical density (IOD) of bands were acquired by image analysis system. Each mRNA expression level was obtained by compared the IOD of each item withβ-actin.1.9 Detecting the expression level of Bcl-2 and Bax protein in HNK treated U937 cells by Western blottingThe neucleoprotein and cytoplasmprotein of HNK treated U937 cells were extracted, and protein concentrations were detected by coomassie brilliant blue G250 kit, applied 100μg protein in each comb, and protein strap were separated by SDS-PAGE gel electrophoresis, Electrophoresis were stopped when bromophenol blue reached the bottom of separation gel. According to the standard protein bands, PAGE gels were cut off and transferred to nitrocellulose membrane, blocked in 5% bovine serum albumin (BSA). Membrane was incubated with the primary antibody at 4℃for overnight. The membrane was washed three times with TBS, then added secondary antibody labeled with horseradish peroxidase (HRP), incubated at 37℃for 1 hour, washed three times again. Protein expression was detected with chemiluminescenc and the band IOD was measured with UVP lab work software.2 Establishing an Adriamycin (ADR)-resistant human acute leukemia cell line U937/ADR2.1 Inducing the resistance of U937 cell line to ADRThe U937 IC50 to ADR was determined by MTT analysis and calculated by IC50 softwear. U937 cells at Logarithmic growth phase were treated with ADR at the concentration of IC50 (0.85μg/ml), 2 hours later, the cells were transferred to ADR-free medium. Most of cells died, and the survived cells grew slowly. When survived cells entering logarithmic growth phase, these cells were treated with ADR again repeatedly as mentioned above. Every 4 week, MTT method was used to measure the growth inhibition rate until the cells developed the resistance to ADR and its resistance character kept stable in drug-free medium. After 32 weeks'treatment with ADR, the ADR resistant U937 cell line was established, and this cell line was given the name of U937/ADR. The biological characters of U937/ADR were analyzed as following.2.2 Observing the growth curve and the cloning efficiency of U937/ADR U937/ADR cells, at logarithmic growth phase and at the density of 1×104, were seeded into 6-well plate. Cells were count every day for 7 days. Population doubling time was calculated according to Patterson formula. 200 of U937 or U937/ADR cells were seeded into 24-well plate spread with methyl cellulose soft agar medium. Coloning efficiency=colony number/feeding number of cells×100%.2.3 Analyzing Cell cycle distribution of U937/ADR cells by FCMU937/ADR cells were fixed with 70% alcohol, stained with 1ml (50μg/ml) propidium iodide for 30 min, filtered with 35mm nylon mesh, the DNA content and cell cycle distribution was analyzed by flow cytometry.2.5 Analyzing the multidrug resistance spectrum by MTT method U937/ADR were seeded in 96-well plate and treated with different concentrations of chemotherapy drugs (VP-16, Pirarubicin, Vincristine, Mitoxantrone and cytosine arabinoside) for 24 hours. The growth inhibition rates were measured with MTT method, the IC50 of each drug to U937/ADR was calculated by an IC50 calculating softwear. The resistant index was obtained by comparing IC50 (U937/ADR) with IC50 (U937) for each drug.2.6 Detecting the expression level of P-gp, MRP1, LRP, BCRP, GST-π, TopoII, Bcl-2 and Bax proteins in U937/ADR cells by Western blotting. See the above mentioned methods.3 The growth inhibition effect of honokiol on U937/ADR and its mechanism of anti-multidrug resistance3.1 Detecting the IC50 and IC20 of HNK to U937/ADR cells U937/ADR cells were seeded in 96-well plate, treated with different concentrations of HNK for 24 hours, then the cells was added MTT and cultured for another 4 hours. Before MTT assay, the supernatant medium was discarded, DMSO added, and finally the absorbance value (A value) of each well was measured at 570nm. The IC50 and IC20of HNK to U937/ADR was calculated by an IC50 calculating softwear.3.2 The growth inhibition effect of honokiol combined with ADR on U937/ ADR cellsU937/ADR cells were treated with the confirmed IC20 of HNK and different concentrations of ADR for 24 hours, then the growth inhibition rate of U937/ADR cells from each treatment group was measured by MTT method. The anti-leukemia effect of two drugs was calculated according to the following formula: Q = (Ea+b)/(Ea+Eb-Ea×Eb). Ea represents the growth inhibition rate induced by HNK, Eb epresents the growth inhibition rate induced by ADR,Ea+b epresents the growth inhibition rate induced by HNK combined with ADR.3.3 detecting the effect of HNK on P-gp's function in U937/ADR cell Rh123 accumulation method: the density of 5×106/ml of U937/ADR and U937, incubated with 0μg/ml and 6.5μg/ml HNK for 1h and 12h, then Rh123 added for 1h, finally FCM detected the fluorenscence intensity of Rh123 in cells.3.4 The mechanism of drug resistance-reversing of HNK3.4.1 Detecting the activated NFκB in U937/ADR's nucleus with ArrayStarTM NF-κB Kit.3.4.2 Detecting the effect of HNK on the expression of NF-κB/p65 and P-gp protein in U937/ADR cells, after the treatment with HNK or ADR or both of them, by the methods mentioned above. Results1 The honokiol inhibited the growth of U937 leukemic cells.1.1 MTT assay showed that Honokiol could significantly inhibit the proliferation of U937 cells but slightly inhibit the proliferation of PBMCs. The anti-proliferative effect of HNK was in a time- and dosage-dependent manner1.2 Flow Cytometry assay showed that most Honokiol-treated cells were arrested at G0/G1 phase, and the cell number in G2/M and S phase decreased (P<0.05).1.3 Cell morphological change observed under Transmission Electron Microscope revealed that Honokiol-treated U937 cells showing chromatin condensation, nuclear fragmentation and the formation of apoptotic bodies.1.4 FCM analysis demonstrated that Honokiol could induce apoptosis of U937 cells in a time and concentration manner.1.5 The change of the activity of caspase-3After treated with HNK, the ratios of the caspase-3 activity in HNK treated U937 cells to that in control group were: 1.94±0.05,3.59±0.31,11.91±0.17,indicating that the caspase-3 activity increased significantly in HNK treated U937 cells in a time dependent manner.1.6 The effect of mitochondrial permeability transition pore inhibitor on the growth of U937 cells The growth inhibition rates of U937 cells, treated with HNK at the concentration of 10μg/ml for 6h or 12h, was 8.45±0.35% and 31.25±1.30%, respectively. The inhibition rates of U937 cells, treated with HNK at the concentration of 10μg/ml together with CsA for 6h or 12h, was 3.14±0.51%,19.39±0.92, respectively, indicating that HNK's anti-leukemia effect might be via mitochondrial permeability transition pore.1.7 RT-PCR method showed that the mRNA expression of bax increased significantly in U937 cells but not in PBMCs after the cells treated with honokiol,While the expression of bcl-2 mRNA had no significant change.1.8 Western blotting method demonstrated that the protein expression of bax increased significantly in U937 cells after the treatment with honokiol. The expression of bcl-2 protein had no significant change.2 Establishment and characterization of ADR-resistant human acute leukemic cell line, U937/ADR2.1 Establishment of U937/ADR cell line U937 cells were cultured with exposure to high dose (IC50: 0.85μg/ml) of ADR periodically (2 hours). After 5 months'treatment and 51 passages, an ADR-resistant U937 cell line was established, named as U937/ADR. The IC50 of ADR to U937 was 0.85μg/ml and that of U937/ADR was 9.35μg/ml. The ADR resistant character of U937/ADR kept stable in drug-free medium for more than 2 weeks.2.2 The bio-characteristics of U937/ADRWhen cultured in common medium, the population doubling time of U937 cell was 34.7 hours, and that of U937/ADR cell was 43.6 hours which prolonged 8.9 hours. When cultured in ADR (0.85μg/ml) medium, the population doubling time of U937/ADR cell was 46.9 hours, but almost all of U937 died after 72 hours; There was no difference in cloning efficiency between U937 and U937/ADR.2.3 Distribution of cell cycleCompared with of U937 cell, the G0/G1 phase of U937/ADR increased and G2/M decreased, but had no difference in the distribution of S phase. 2.4 The multdrug resistant spectrum of U937/ADRThe resistant index of U937/ADR to ADR is 11, to VP-16 6.5, to Pirarubicin 13.4, to Vincristine 6.1, to Mitoxantrone 4.8, and U937/ADR still kept sensitive to cytosine arabinoside.2.5 The expression of multidrug resistant related proteins in U937 and U937/ADR cellsThe multidrug resistant related proteins, such as P-gp, LRP, GST-π, Topo II, Bcl-2 and Bax in were expressed at low levels in both U937 and U937/ADR cells. The expression of P-gp in U937/ADR was significantly higher than that in U937. MRP1 and BCRP were not detected in both U937 and U937/ADR cells.3 The effect of honokiol on reversing multidrug resistance in U937/ADR cells3.1 The IC50s of HNK to U937/ADR IC50 of honokiol to U937/ADR was 14.3μg/ml, and IC20 was 6.5μg/ml.3.2 The IC50s of ADR to U937/ADR after the cells treated with HNK The IC50 of ADR to U937/ADR without HNK treatment was 9.35μg/ml, while the IC50 of ADR to U937/ADR after HNK treatment decrease to 4.28μg/ml3.3 The synergistic effect of honokiol combined with ADR on U937/ADR cellsU937/ADR cells were treated with HNK for 24h, washed twice, then added ADR for another 24h's culturing. The cell growing inhibition detecting results showed there was a synergistic effect of HNK combined with ADR.3.4 HNK could improve the Rh123 accumulation in U937/ADR after treated with HNK for 12h, but not 1h.3.5 The mechanism of drug resistance-reversing of HNK3.5.1 The activated NF-κB measurement result showed that in the activated NF-κB in U937/ADR nucleus was p65 subunit, not p50.3.5.2 The effect of HNK on the expression of NF-κB/p65 and P-gp proteinThe present study result demonstrated that HNK reduced the activation and expression of NFκB/p65 protein and decreased the expression of P-gp protein.Conclusions1. Honokiol could inhibit the proliferation and induce apoptosis of U937 cells, while had little effect on the proliferation and survival of PBMCs. Bax might be involved in the gene regulation related to Honokiol-induced apoptosis.2. The U937/ADR cell line was established by exposing U937 cells to high dose of ADR periodically, and U937/ADR's resistant index to ADR was 11. In addition, U937/ADR had drug resistance to other chemo-therap- eutics drugs, such as VP16, VCR, THP and MIT, which indicated U937/ADR cell line was a typical MDR cell line.3. HNK could decrease the protein expression of P-gp in U937/ADR, and increase the ADR distribution in U937/ADR. Therefore, the drug resistance of U937/ADR to ADR would be reversed by HNK to some extend.4. HNK could inhibit the activation of NF-κB/p65, and decrease the P-gp expression.5. HNK might be an effective anti-leukemia drug.