Honokiol augments the anti-cancer effects of oxaliplatin on colon cancer cell : Apoptosis and analysis of the molecular mechanisms

The drug oxaliplatin is important in the chemotherapy of colorectal carcinoma, but its toxicity, especially dose-related neurosensory toxicity, is not well tolerated. We investigated if honokiol could augment the anti-tumor effect of oxaliplatin in colon cancer HT-29 cells in vitro and determined if honokiol could be used with oxaliplatin to decrease its dose. Cell proliferation, apoptosis, and prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF) levels were investigated. Expression of cyclo-oxygenase 2 (COX-2), VEGF, AKT/p-AKT, extracellular signal-related kinase (ERK)1/2/p-ERK1/2, nuclear factor kappa B (NF-κB), P65/p-P65, and caspase-3 was examined. Honokiol or oxaliplatin alone suppressed the proliferation of HT-29 cells in a concentration-dependent manner. HT-29 cells were more sensitive to oxaliplatin treatment in the presence of honokiol. Oxaliplatin combined with honokiol improved the rate of HT-29 cell apoptosis and reduced PGE2 and VEGF secretion levels. Expression of COX-2 and VEGF protein and phosphorylation of AKT, ERK1/2, NF-κB and P65 were also inhibited, caspase-3 levels were upregulated after honokiol treatment. Therefore, honokiol can be combined with oxaliplatin in the chemotherapy of colorectal carcinoma, this combination allows a reduction in oxaliplatin dose, and thereby reduces its adverse effects, and may also enhance the chemotherapeutic effect of oxaliplatin for this disease.


INTRODUCTION
Oxaliplatin is a platinum-based chemotherapeutic agent with a 1,2-diaminocyclohexane carrier ligand that produces bulkier DNA conjugates due to the restricted freedom of motion of the platinum atom.Oxaliplatin plays very important role in the chemotherapy of colorectal and ovarian cancer (Kweekel et al., 2005).The chemotherapy regimens, folinic acid/fluorouracil/oxaliplatin (FOLFOX) or capecitabine/oxaliplatin (XeLOX) are a first-line treatment in advanced colorectal cancer.Oxaliplatin combined with fluorouracil (5-Fu) can markedly improve the 5-year survival rates of colorectal cancer patients, but oxaliplatin toxicity, especially its dose-related neurotoxicity (Cavalettia et al., 2001;Pasetto et al., 2006), is not well tolerated by most patients.Drug resistance to oxaliplatin is also a problem in chemotherapy.Therefore, finding the right dosing scheme and strategy for each individual patient that minimizes the side effects remains a challenge for individual-based chemotherapy management.Meanwhile, the discovery of new drugs that can augment the anti-tumor effect of oxaliplatin efficiently and also enable a reduction in its dose is needed urgently.
Honokiol is an active component that has been isolated and purified from the Chinese traditional herb magnolia.It has been shown to have anti-angiogenic, anti-invasive, and anti-proliferative activities in several types of human cancer cells (Han et al., 2009), which include leukemia (Hibasami et al., 1998;Battle et al., 2005), human breast cancer cells (Liu et al., 2008;Park et al., 2009), human hepatocellular (Han et al., 2009), human multiple myeloma (Ishitsuka et al., 2005), human prostate cancer cells (Hahm and Singh, 2007), and human squamous lung cancer (Yang et al., 2002).In this study, we analyzed the effect of honokiol, either alone or in combination with oxaliplatin, on the proliferation and apoptosis of the human colon cancer cell line HT-29.We also investigated the expression of several downstream molecular mechanisms to analyse the way in which honokiol may induce cell apoptosis.

Annexin V/propidium iodide (PI) apoptosis assay
Cell apoptosis was measured using an annexin V-FITC apoptosis detection kit (BD PharMingen, CA, USA).Briefly, HT-29 cells were removed from the culture dish and stained with annexin V-FITC and PI and were analyzed by flow cytometry (FACSCalibur, BD PharMingen) after treatment.Cells that were annexin V-FITC and PI double-negative were considered to be non-apoptotic for statistical analysis.

Analysis for prostaglandin E2 (PGE2) and VEGF production
The concentrations of PGE2 and VEGF in culture supernatants were determined using a competitive enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, MN, USA).

Western blotting
Cells were treated with honokiol or oxaliplatin alone or combined, then harvested and washed three times with ice-cold phosphatebuffered saline (PBS).Cell lysates were prepared for western blot analysis of VEGF, COX-2, GAPDH using whole cellular protein extraction kits (Active Motif, California, USA).A DC protein assay kit was used (Bio-Rad, Richmond, CA, USA) to examine the protein concentration in each cell lysate; 40 µg protein was mixed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer and denatured for 10 min at 95°C.Proteins were separated on a 10% polyacrylamide gel and blotted on a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA).Nitrocellulose membranes were blocked with 5% bovine serum albumin (BSA; Sigma) in Tris-buffered saline (TBS; 25 mM Tris-HCl, 150 mM sodium chloride and pH 7.2) for 2 h at room temperature.Blots were incubated with rabbit polyclonal immunoglobulin G (IgG) primary antibody overnight at 4°C.Blots were washed three times with washing buffer (PBS with 0.1% Tween-20) and then incubated in horseradish peroxidase (HRP)conjugated goat anti-rabbit second antibody (1:2000 dilution) for 2 h at room temperature.After thorough washing, the blots were incubated with HRP-conjugated secondary antibody.The reaction was developed using enhanced chemiluminescence (ECL) reagents (Amersham, NJ, USA) and analyzed using a VersaDoc MP5000 imaging system (Bio-Rad, CA, USA).

Statistical analysis
The results were expressed as the mean value and standard error of the mean.Statistical significance was analyzed by one-way analysis of variance (ANOVA).A value of P < 0.05 was considered to be statistically significant.

Effect of honokiol and oxaliplatin on inhibition of proliferation of HT-29 cells
We used the 3-(4,5-dimethythiazol-.2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to detect HT-29 cells viability after treatment with different concentrations of honokiol or oxaliplatin.The addition of either honokiol or oxaliplatin had a concentration-dependent inhibitory effect (Figure 1).A honokiol concentration of 20 µM was the maximum concentration that did not affect HT-29 cell proliferation and oxaliplatin at 0.6 µM was the minimum effective concentration.The anti-proliferation capability of oxaliplatin was enhanced significantly when honokiol was also added to the cells (Figure 2).The data showed that HT-29 cells were more sensitive to the combined treatment than treatment with single reagents alone.The addition of honokiol markedly increased the antiproliferation effect of low concentrations of oxaliplatin.

Effect of honokiol and oxaliplatin on induction of apoptosis of HT-29 cells in vitro
A range of concentrations of honokiol (0, 0.2, 1, 5, and 20 µM) combined with 0.6 µM oxaliplatin was used.The percentage of apoptotic HT-29 cells increased significantly when honokiol was added (Figure 3); for example, the addition of honokiol (20 µM) increased the number of cells that were undergoing apoptosis from 9.61 to 44.22%.Therefore, there was a significant synergistic effect following honokiol and oxaliplatin treatment.The induction of cell apoptosis was more effective at a lower concentration of oxaliplatin in the presence of honokiol.

PGE 2 and VEGF production in culture supernatants
The levels of PGE 2 and VEGF in HT-29 cells culture supernatants were examined by competitive ELISA after treatment with honokiol or oxaliplatin alone or combined.
Addition of honokiol at concentrations greater than 1 µM reduced the production of PGE 2 and VEGF (Figure 4) in a concentration-dependent manner.Honokiol added at concentrations above 5 µM had a significant suppressive effect when compared with the control group, independent of the addition of oxaliplatin (P < 0.01).There was a synergetic suppressive effect between oxaliplatin and honokiol when honokiol was at added at concentrations between 1 to 5 µM (P < 0.05).

Possible mechanisms of honokiol induction of HT-29 cell apoptosis
We found that honokiol combined with low concentrations of oxaliplatin (0.6 µmol/L) suppressed HT-29 cell proliferation and induced apoptosis markedly; therefore, we investigated the possible mechanisms of honokiolinduced HT-29 cell apoptosis using western blotting.
Honokiol at a concentration of 20 µM reduced the production of VEGF and COX-2 proteins significantly, inhibited the phosphorylation of AKT, ERK1/2 and NF-κB P65, and caspase-3 expression was upregulated (Figure 5).This effect was increased if oxaliplatin and honokiol were given together.However, there was no effect of addition of oxaliplatin alone when compared with the control group.

DISCUSSION
Oxaliplatin therapy has been considered to be a first-line therapy strategy in the chemotherapy of advanced colorectal cancer.But oxaliplatin toxicity, especially its neurotoxicity, is not well tolerated by most patients.After long-term administration of oxaliplatin, patients may present with deep sensory loss, sensory ataxia and functional impairment.This type of neurotoxicity usually has late onset and is correlated with the cumulative dose of oxaliplatin.Resistance to oxaliplatin is also a problem after long-term therapy.Severe toxic reactions and high resistance to this drug after long-term treatment limit its clinical application, therefore, there is an urgent need to minimize the adverse effects and improve the anti-cancer functions.
In recent years, anti-cancer agents derived from natural products have been considered to play an important role in the development of cancer therapy.Honokiol is a neolignan isolated from the traditional medicinal herb Magnoliae cortex, it has been shown to be effective in the therapy of several types of human cancer cells, such as breast cancer, human hepatocellular carcinoma, leukemia, human prostate cancer cells, human squamous lung cancer, and human multiple myeloma.Honokiol can traverse the blood-brain barrier and induce apoptosis of neuroblastoma (Lin et al., 2012).Honokiol was also observed to have antimetastatic activity in osteosarcoma (Steinmann et al., 2012).In this study, we evaluated the anti-cancer value of honokiol in colon cancer HT-29 cells.We found that low concentrations oxaliplatin combined with non-toxic concentrations of honokiol had a much more powerful effect on inhibition of cell proliferation, induction of apoptosis, and inhibition on PGE 2 and VEGF expression in human colon HT-29 cells than either oxaliplatin or honokiol alone.We also investigated the molecule mechanisms of honokiol induction of cell apoptosis.Honokiol could suppress the expression of VEGF and COX-2, inhibit the phosphorylation of AKT, ERK1/2 and NF-κB P65, and upregulate the expression of caspase-3.
NF-κB plays a major role in the control of apoptosis, cell proliferation and differentiation, and is activated in response to several pro-apoptotic stimuli, such as tumor necrosis factor (TNF)-α, ionizing radiation, oxidative stress, and cytotoxic.NF-κB phosphorylated by these stimuli then translocates into the nucleus and regulates the expression of anti-apoptotic genes.Therefore, inhibition of NF-κB in cancer cells has become one of the major targets of anti-cancer therapy.
These studies suggest that honokiol could induce cell apoptosis and inhibit cell proliferation by the suppression of AKT, ERK1/2, and NF-κB P65 phosphorylation, by the suppression of COX-2 and VEGF expression, and by the upregulation of caspase-3 expression.These results show, as far as we know for the first time, that honokiol can augment the anti-tumor effect of oxaliplatin.This effect may not only enable a reduction of the dose of oxaliplatin given to patients and thereby prevent the associated adverse effects, but may also enhance the chemotherapeutic effect on colon cancer.Honokiol can reduce the toxicity and side effects of oxaliplatin by decreasing the dosage, and lead to improved efficacy and less drug resistance of oxaliplatin in chemotherapy.Patients who cannot stand the oxaliplatin toxicity or have poor effect will benefit from honokiol.

Figure 5 .
Figure 5.Western blood analysis of protein cell lysates to investigate a possible mechanism of honokiol induction of HT-29 cells apoptosis.GAPDH was used as the positive control.