Key words】 colorectal bcl  2 bax paeonol

    Chemotherapy drug resistance [1] and its side effects has been the two major problems of the treatment of colorectal cancer, and to find effective and side effects of anticancer drugs at home and abroad has become an important research topic in the plants. Studies have shown to have some anti-tumor activity of paeonol (Paeonol, Pae), intragastric administration with anti-mouse liver tumor effect [2]. We have reported Pae significantly inhibited the in vitro proliferation of human colorectal HT  29 cells [3]. To further explore the possible mechanism of this inhibition, we carried out the following research, for Pae clinical applications provide a theoretical and experimental basis.

    1 Materials and methods

    1.1 Cell culture

    The conventional culture of HT  29 cells, take the logarithmic growth phase cells used in the experiment. The cells were divided into experimental and control groups in the experimental group were added to the the different concentration Pae solution (as 15.63mg / L, respectively, 62.5mg / L, 250 mg / L), the control group, added the same amount of culture medium.

    1.2 Pae induced apoptosis

    1.2.1 observed under an inverted microscope the conventional to flasks training of HT  29 cells, inverted microscope continuity dynamic observation cell growth.

    1.2.2 TUNEL assay of apoptosis cells home there coverslip seeded in 6-well culture plate, adherent divided into experimental and control groups after 48 hours, remove the coverslip treatment by TUNEL kit instructions. Light microscope observations nucleus stained tan convicted of apoptotic cells, five randomly selected high magnification (× 200) field of view, counting 200 cells per field, apoptotic index (AI) = apoptotic cell number / the total number of cells × 100%.

    1.2.3 Flow cytometry cytometry cell cycle digestion cells collected in each experimental group and the control group cultured 48h, centrifuged, rinsed, filtering, fixed, adding RNase and PI staining detected on the machine.

    1.3 apoptosis related gene bcl  2, bax detection

    The cells were seeded and packet TUNEL method, 48h after rinsing, fixed, dropping an anti-(bcl  2 and bax antibody) as a negative control antibody was replaced with PBS, I step SP kit instructions. Each slide in a 40 × 10 high power microscope observe cells stained tan sentenced positive cells stained negative cells. Apoptosis gene associated protein bcl  2, bax located in the cytoplasm and membrane. Per field were calculated: expression rate (%) = (number of positive cells / total number of cells) × 100% per smear observed five fields.

    1.4 statistical treatment

    SPSS10.0 statistical software.

    2 Results

    2.1 Pae induced apoptosis of HT  29

    2.1.1 inverted microscope observation vigorous growth control cells, showed high refractive index, large cell body. PAE cells proliferation slows down, with the drug concentration is increased and prolonged, the cell gradually becomes small, the refractive index decreased, partial peeling floating in the culture flask, but intact membranes, and finally cleavage.

    2.1.2 TUNEL the method experimental group apoptotic cells brown stain particles located in the nucleus, positive staining of the cell nucleus fragmentation, nuclear pyknosis, membrane prominent form a plasma membrane vesicles apoptotic morphological changes (Figure omitted). Pae handle 48h HT  29 cell lines the number of apoptotic cells increased significantly, and the differences were statistically significant (P <0.01), and the the AI ​​and Pae concentration was positive phase dependencies are shown in Table 1. Table 1 groups comparison of the AI

    2.1.3 flow cytometry to detect different concentrations the Pae role in HT  29 cells 48h, the cell cycle distribution changed significantly, the performance is the increase in the proportion of cells in S phase G0/G1 phase and G2 / M phase cells were decreased, see Table 2, and the apparent apoptotic peak as shown in Figure 1a to 1d. Compared to the two sets of results were significant differences (P <0.05).

    2.2 Pae apoptosis related gene bcl  2 of bax expression are shown in Table 3. Table 2 Pae HT  29 cell cycle

    2.2.1 expression of bcl  2 protein expression in the control group bcl  2 protein expression level of the highest number of cytoplasmic staining cells up and was dark brown the Pae cells bcl  2 expression were decreased, and the decrease in positive cells, staining was significantly lighter inversely proportional relationship, and the drug concentration in each group the results were compared with control group having a significant difference (P <0.01) (figure omitted).

    2.2.2 bax protein positive cytoplasmic membrane dyed dark brown. 0.05) (图略)。">Pae group staining positive cell count and the degree of staining with the control group showed no significant difference (P> 0.05) (Figure omitted).

    3 Discussion

    Excessive cell proliferation and apoptosis inhibition is considered to be the key to tumor development [4]. Numerous studies have shown that the reduction of apoptosis and the incidence of colorectal cancer development related. Variety of chemotherapy drugs can cause tumor cell apoptosis [5]. The treatment of cancer by inducing apoptosis is currently a hot topic [6]. The Pae role in the experimental colorectal cancer HT  29 cells, typical apoptotic morphological changes can be observed through the light microscope. The TUNEL method colorectal cancer cell apoptosis index increased significantly after Pae processing dose-dependent manner, and with Pae. Obvious apoptotic peak flow cytometry found Pae role, the cell cycle distribution significantly change Pae HT  29 cell cycle distribution block the cell cycle in the S phase to the G2 / M phase transition process of to reduce mitosis, and induce apoptosis. Confirmed Pae role in the mechanism of action of the HT  29 cells and induced apoptosis of the cell lines and related to the distribution of the cell cycle.

    Apoptosis is a programmed process of multi-gene regulation. By studying the molecular regulation mechanism, will plan to induce tumor cell apoptosis important role in guiding [7]. Bcl  2 family, p53, Fas, c-myc, k  ras and other genes associated with apoptosis broadly, including bcl  2 is the central link of apoptosis regulation. In vitro experiments showed that the addition to the growth factor, the normal cells will gradually steering apoptosis; when bcl  2 overexpression, apoptosis is inhibited [8]. Thus, bcl  2 may prolong cell life by inhibiting apoptosis, the produce excessive cell proliferation and accumulation, use start action tumorigenesis. bax genes are new discoveries in recent years, an apoptosis-promoting genes are bcl  2 of the same family [9]. bax gene bcl  2 In contrast, the monomer and bax / bax forms homodimers both pro-apoptotic role. bax can be bcl  2 to form heterodimers promote apoptosis, inhibition of the function of the latter. Recent studies have shown that bcl  2 family to promote a balance between apoptotic and anti-apoptotic proteins in the regulation of pro-apoptotic factor c  myc release from the mitochondria play an important role [10]. The protein bcl  2/bax proportion is a key factor in the occurrence or non-occurrence of apoptosis [11]. The experiment found Pae significantly lowered colorectal HT  29 of bcl  2 gene expression of bax after Pae role compared with the control group, the expression rate was no significant difference, it may be that after Pae role bcl  2 / Bax decline in the proportion of colorectal cancer cells, thereby inducing apoptosis.

    In summary, this experimental study found that The TCM Paeonol in certain concentration range can be significantly lowered colorectal carcinoma HT  29 cells expression of bcl  2/bax proportion, Therefore, the paeonol the inhibitory mechanism may be one bcl  2 and bax genes to induce apoptosis of tumor cells by acting on.