Abstract Objective To investigate paeonol and adriamycin inhibited the proliferation of HepG2 cells. Methods MTT assay determination of paeonol, doxorubicin and paeonol combined with doxorubicin in vitro on HepG2 cell proliferation inhibition rate; evaluation of the interaction between the two drugs index nature of the interaction of the two drugs; flow cytometry to detect apoptosis ; morphological changes of cell apoptosis in situ detection of apoptosis were observed; Western blot analysis of Bcl  2, Bax changes. Results with different concentrations of paeonol, doxorubicin, and the combination of HepG2 cells were proliferation inhibition in a dose-dependent manner. Paeonol 31.25mg / L, respectively, with doxorubicin in 0.16,0.31 mg / L when combined synergistic effect is most significant (CDI <0.7). Flow cytometry apoptosis rate of the combined treatment group (37%) was significantly higher than that in the control group and the medication alone group. TUNEL staining showed typical apoptotic morphological features. Western blot analysis showed that the drug alone and the combination group Bcl  2 expression reduce, Bax upregulation, Bcl  2/Bax ratio decreased. Conclusion certain concentration of paeonol can enhance doxorubicin inhibited the proliferation of HepG2 cells in vitro, its mechanism of action by reducing the expression of Bcl  2 expression of Bax expression, making the Bcl  2/Bax ratio decreased induction of apoptosis achieve ‘s.

Key words paeonol doxorubicin HepG2 cell proliferation and apoptosis

   Hepatocellular liver cancer (hepatocellular carcinoma, HCC) is one of the common malignant tumors in China, most patients are diagnosed at late stage, lost the chance of operation. Doxorubicin is the first-line treatment of liver cancer chemotherapy, however, less than 20% efficiency, and hematologic toxicity and gastrointestinal reactions [1]. Therefore, efforts to find effective drugs and treatment optimization is the focus of the current study.

    Paeonol (paeonol, Pae), also known as paeonol, the main active ingredient Ranunculaceae plant peony root bark and Asclepiadaceae plants Cynanchum dried roots or the whole plant. The study showed that paeonol not only inhibit the proliferation of a variety of tumor cell lines have, but also can enhance cisplatin inhibition of HepG2 cells and the proliferation of SMMC  7721 [2  4].

    In this study, by observing of paeonol and adriamycin inhibit the proliferation of HepG2 cells, and to explore its possible mechanism to provide a theoretical basis for the clinical treatment of liver cancer.

    1 Materials and methods

    1.1 Drugs and reagents

    Pae injection was purchased from Shanghai First Pharmaceutical Factory; hydrochloride for injection doxorubicin Star (doxorubicin, Dox) were purchased from Shenzhen Wanle Pharmaceutical Co., Ltd.; the DMEM culture powder, fetal bovine serum, trypsin was purchased from the United States Hyclone; four MTT (MTT) and mouse anti-human Bax, Bcl  purchased from Sigma, USA; TUNEL purchased in Promegar; BCA protein assay kit provided by Pierce company.

    1.2 Main equipment

    Nacpo  6100 CO2 incubator (DuPont); the YJ  1450 type Medical Clean Bench (Suzhou Purification Equipment Company); inverted microscope (OLYMPUS); EL301 Strip reader (microplate reader) (U.S. BW  Tek) ;

    1.3 cell line

    Human hepatoma cell line HepG2 were purchased from Shanghai Liver Cancer Institute.

    1.4 Experimental Methods

    1.4.1 Preparation of cell cultures and cell suspension HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum, and placing 37 ° C, 5% CO2 incubator, taking the logarithmic growth phase cells used in the experiment.

    1.4.2 tumor cell proliferation in vitro inhibition experiments [5] using the MTT assay, the the set experimental group, the cells in the control group and blank control group. Each hole of the experimental groups were added to 200μl culture was diluted drugs the (PAE final concentration 7.81,15.63,31.25,62.50,125.00,250.00 mg / L, Dox the final concentration for 0.16,0.31,0.63,1.25,2.50 mg / L 1:1, the ratio of the two drugs program), the cells in the control group and blank control group only added to the culture medium of the same amount, with 570nm was measured in a microplate absorbance value (A). The following formula of the drug on tumor cell growth inhibition rate: tumor cell growth inhibition rate (IR) = (1 – experimental group A value / cell control group A value) × 100%. Drug concentration of horizontal axis, and the inhibition rate ordinate plotted growth inhibition curve. Linear regression to the logarithm of the drug concentration and inhibition rate and the obtained IC50 of.

    1.4.3 drugs combined effects evaluation [6] The interaction between the two drugs index (coefficient of drug interaction, CDI) evaluation and nature of the interaction of the two drugs, CDI calculated according to the following formula: CDI = AB / (A × B). According to the number of viable cells (absorbance values) were calculated, AB is the combination of two drugs with the control group the ratio of absorbance values; A or B is a drug used alone with the control group the ratio of absorbance values. 1则两药作用性质为拮抗。">The nature of the role of the two drugs when CDI <1 synergies; synergy is very significant when CDI <0.7; CDI = 1, the role of the nature of the two drugs is additive; the CDI> 1, the role of the nature of the two drugs antagonism.

    1.4.4 Flow cytometry detection of apoptosis and cell cycle changes will collect untreated and drug treatment of liver cancer cells in a test tube (cell number × 105 ~~ 1 × 106), 1 200r/min centrifugal 10min, abandoned on clear, rinsed with PBS 2 times, according to DNA  Prep  Reagents Kit manual operation, are then added to the membrane perforation agent, ethidium bromide (propidium iodide, PI) and RNA staining, dark at room temperature placed 30min, through a 400 mesh screen , flow cytometry, and the excitation wavelength of 488nm to determine the cell cycle and apoptosis rate.

    1.4.5 situ detection of apoptosis (TUNEL) cells were seeded in 6-well plates (including poly-L-lysine-treated coverslips), set to 37 ° C, 5% CO2 incubator cultured overnight, drug remove the cells seeded after 48h. 4% paraformaldehyde 25min TUNEL manual operation, DAB color, contrast staining the dehydration transparent Fengpian observed. The experimental set of positive and negative control at the same time, ordinary light microscope, five randomly selected high power field, counting 000 cells to calculate the value of the percentage of positive cells of the tumor cells, as apoptosis index (apoptosis index, AI).

    1.4.6 Western blot to detect expression of Bcl-2/Bax expression level of cells treated with the drug after 48h, washed twice with ice-cold PBS, adding RIPA buffer solution (150 mmol / L NaCl, 1% NP  40,0.5% deoxycholic acid, 0.1 % SDS, 2mmol / L EDTA, 50mmol / L Tris, pH 8.0) homogenate extracted protein, the BCA Protein Assay, by adding sample buffer, SDS  PAGE electrophoresis, the protein content is about 40μg / hole, the protein transferred to PVDF membrane, Ponceau S staining, containing 5% nonfat dry milk in PBS closed 2h, were added to the mouse anti-human Bcl-2, Bax, 4 ° C were incubated overnight, PBS washing the membrane to the corresponding HRP-labeled sheep anti-mouse IgG, 37 ℃ incubated for 2h ECL color bands shades was observed and analyzed as a control, β  actin.

    1.5 statistical methods

    Application SSPS10.0 software for statistical analysis. The ± s Kruskal  Wallis rank sum test and Pearson correlation statistical analysis, P <0.05 for the difference was statistically significant.

    2 Results

    2.1 paeonol and doxorubicin alone inhibited the proliferation of HepG2 cells

    Pae in the range of 7.81 ~ 250mg / L concentration of HepG2 cells proliferation inhibitory rates with the drug concentration liter

    Increase Pae 7.81,15.63,31.25,62.50,125.00,250.00 mg / L concentration of HepG2 inhibition rate as follows: 0.93%, 14.21%, 16.87%, 40.13%, 78.69%, 98.79%. Linear correlation analysis to the logarithm of the concentration and the inhibition rate: r = 0.9637, P <0.01. The IC50 was 58.943 ± 2.457.

    Series of concentrations of Dox (0.16,0.31,0.63,1.25,2.50 mg / L) alone on the proliferation of HepG2 cells was inhibited, the inhibition rate of 18.36%, 33.53%, 61.55%, 74.89%, 77.03%. The higher the concentration of the drug, the stronger the inhibition, showing a significant dose-dependent effect relationship. Linear correlation analysis to the logarithm of the concentration and the inhibition rate: r = 0.9643, P <0.01. The IC50 of 0.543 ± 0.097.

    Of 2.2 Paeonol and Adriamycin inhibited the proliferation of HepG2 cells

    The combined effect of three concentrations Pae (15.63,31.25,62.50 mg / L) with different concentrations of Dox in HepG2 cells can be observed and their synergistic proliferation inhibition, Figure 1. The CDI analysis showed that, when combined with Pae DOX 0.16,0.31 mg / L, i.e. display a synergistic effect (CDI <1). Which Pae 31.25mg / L combined synergies of the most significant (CDI <0.7), the CDI were 0.656,0.635, compared to the combined treatment group with the same dose of Dox single drug group difference was statistically significant (P <0.01 ). However, when Dox concentration 0.63,1.25 mg / L, antagonism, combined with 31.25mg / L Pae CDI were 0.930,1.097; 62.5mg Pae associated with CDI order 1.047 1.304, see Figure 2.

    The 2.3 Paeonol apoptosis-inducing effect of doxorubicin

    Appear before the G1 phase of the sub-G1 represents the number of apoptotic cells. FCM analysis showed that the administration group than in the control group, showing significant sub-G1 phase, in which the most significant apoptosis peak of the combined group, the apoptosis rate of up to 37%, the same compared to the control group and the single drug group P < 0.01, see Figure 3.

    The typical apoptotic performance chromatin condensation, edge set, crescent-shaped gathered at the nuclear membrane. Agglutination chromatin different forms due to the different aspect scattered in an arbitrary position of the nuclear aspect, or a round is located in the nuclear membrane, or is located in the core central. TUNEL staining of a little nucleus deep tan stained apoptotic cells in the control group. In the the doxorubicin and paeonol single drug group, and the number of apoptotic cells in the combination group in turn increased most significantly to the combined treatment group, as shown in Figure 4.

    2.4 paeonol and doxorubicin Bcl  2 and Bax expression

    Western blot analysis showed that paeonol and doxorubicin alone treatment group, Bcl  2 expression is significantly reduced and Bax expression increases of Bcl  2/Bax the ratio in the administration group have different levels of reduction, the combined treatment group the most significant, as shown in Figure 5.

    3 Discussion

    Liver cancer is one of the most common tumor in China and sub-Saharan Africa region. In recent years, the incidence of a rising trend in developed countries [7]. HCC patients is not sensitive to anticancer drugs, the dose of the drug while chemotherapy by liver function limitations, therefore, did not play an important role in chemotherapy in the treatment of liver cancer. Surgery, arterial embolization and other general therapy in patients with advanced or relapsed, chemotherapy is one of an important alternative method. However, so far, have not found a single agent or combination of its efficiency of 25% [8]. Doxorubicin is a cell cycle non-specific drugs, its direct killing effect by the tumor cell cycle arrest at the G2 / M phase [9]. This study shows that both the proliferation of HepG2 cells in vitro tests of paeonol and doxorubicin combination inhibited in a dose-dependent manner. When given 31.25mg / L paeonol combined with very low concentrations of doxorubicin (0.16 or 0.31mg / L), that showed significant synergy (CDI <0.7), and inhibit the proliferation of HepG2 cells The role was significantly higher than of paeonol, or doxorubicin monotherapy group.

    Many studies have shown that Bcl  2 many of the family members, Bcl  2 gene is the main inhibitor of apoptosis, and Bax activate gene is the most important. Bcl  2 the BH region with Bax is combined the formation of the heterodimers and Bcl  2/Bax ratio plays an important role in the process prevent Bax regulation of apoptosis [10]. Bcl  2 down or the Bax upregulation can promote apoptosis [11]. In the present study, flow cytometric analysis, The paeonol doxorubicin induced apoptosis in HepG2 cells apoptosis rate as high as 37% in the combination therapy can in varying degrees. This phenomenon is also confirmed by TUNEL staining. A large number of apoptotic cells in the administration group, showing significant nuclear uptake and nuclear fragmentation. Apoptosis is particularly evident in the combination group. Western blot analysis showed that, after the administration of Bcl  2 expression down, Bax upregulation, Bcl  2/Bax ratio for the combination group.

    In short, of paeonol and adriamycin on the proliferation of HepG2 cells inhibit synergy, its mechanism may be that by down-regulating expression of Bcl  2 expression of Bax expression Bcl  2/Bax ratio decreased induction of apoptosis, however, the exact mechanisms need to be further studied.