Abstract】 Objective the study p27mt genes inhibit growth of colorectal cancer xenografts in nude mice and antimetastatic effects and mechanisms. Established colorectal cancer xenografts in nude mice, using direct intratumoral injection, measurement of tumor volume, to observe p27mt tumor growth inhibition. Separation of the tumor, and the production of single-cell suspensions, flow cytometry cell cycle, apoptosis was measured. Immunohistochemical detection of tumor tissue expression of MMP 9. Results Ad p27mt group, Ad LacZ group and the control group, xenografts average volume, respectively (1.94 ± 0.67) cm3 (2.75 ± 0.83) cm3 and (3.01 ± 0.76) cm3 (P <0.05); proliferation rate ( 37.34 ± 1.45)%, (53.16 ± 3.27)% and (54.48 ± 2.43)% (P <0.05); apoptosis rates were (19.79 ± 3.32)%, (6.38 ± 4.91)% and (7.25 ± 5.20)% (P <0.01); MMP 9 expression was 20%, 75% and 66.7% (P <0.01). Conclusion mutant p27mt gene by blocking the transplanted tumor proliferation and promote apoptosis of transplanted tumor cells to inhibit tumor growth inhibition of transplanted tumor metastasis by reducing the transplanted tumor expression of MMP 9.
Key words] of colorectal p27mt gene in nude mice MMP 9
With the improvement of people’s living standards, changes in diet, the incidence of colon cancer is a gradual increase in . Treatment of colon cancer include surgery, radiotherapy and chemotherapy, but the results are not satisfactory. With the advent of the post-genome era, become the focus of people study gene function, cancer gene therapy has brought the dawn. The p27 gene is a tumor suppressor gene, this study aimed to explore the mechanism of action of mutant p27 gene on the growth and metastasis of colorectal cancer in animal experiments, and provide the basis for the treatment of colorectal cancer for p27.
1 Materials and methods
1.1 cell lines and adenovirus
99%。">Lovo cells were purchased from Wuhan University Standard Collection and cultured with RPMI1640 medium, adjusting the cell concentration to 1 × 108/ml trypan blue staining live cell count> 99%. Ad LacZ by Dr. Wang Jianing and other building and the kindly . Ad p27mt built by me .
1.2 colon cancer nude mouse model establishment and packet purchase of BALB / C nude mice 36 (Hubei experimental animal tube Table 1 groups transplanted tumor cell cycle distribution, PI and apoptosis rate *: Ad p27mt group comparisons with control group and Ad of p27mt group, P <0.01 processing center), the mice aged 4 to 6 weeks, body weight 18 to 25 g. Take 0.2ml of colon cancer the Lovo cell suspension, respectively, in the right side of the back of nude mice inoculated subcutaneously with a total of 36. Selection of tumor size tumor 27, ranged between 0.5 ~ 1.5cm nude mice were randomly divided into a control group, Ad LacZ group and Ad of p27mt group. Direct intratumoral injection, each were injected with PBS 0.1 ml of Ad LacZ0.1ml (and virus content 1010pfu/ml) and Ad p27mt 0.1 ml of (virus content 1010pfu/ml). 3, 1, 28 days in a row.
1.3 xenograft tumor volume determination
The transplanted tumor was measured the long diameter (a) and the shorter diameter (b), according to the formula V = A2B / 2 calculating the volume.
1.4 Flow cytometry
To be a nude mouse model of treatment 28d, the mice were sacrificed, separation tumor weighed and photographed. Transplanted tumor tissue 15g were cut into single cell suspension, take 100 μl of to join DNA PREPTMLPR 200μl mix, DNA PREPstain 1 000μl staining reagent mix after adding in the dark at room temperature 3min, 15min after Coulter Epics XL flow cytometry The instrument detected on the cell cycle and apoptosis. Cell proliferation index (PI) was calculated according to the following formula:
PI = S + G2/MG0/G1 + S + G2 / M × 100%
1.5 Immunohistochemical detection of the expression of MMP 9
SP staining mouse anti-human MMP 9 monoclonal antibody, SP kit of DAB chromogenic reagent purchased from Beijing Zhongshan specific steps according to kit instructions. Results criteria: The expression of MMP 9 brown granules in the cytoplasm. Matrix in the tumor area, counting five fields per field count the total number of cells and expression of MMP 9 positive number of cancer cells, according to the scope and extent of the cell coloring immunohistochemistry were divided into: – no positive staining cells; + the pale or colorant cells <10%; + + coloring moderate or coloring cells accounted for 10% to 25%; + + + deep coloring or staining cells is greater than 50%. – A negative expression; + / + weak expression; + + + expression was strongly positive.
1.6 statistical methods
Measurement data with the mean ± standard deviation (± s) said, using independent sample t-test, χ2 test of count data.
Final volume of 2.1 tumor xenografts comparison
Ad p27mt group the xenografts average volume (1.94 ± 0.67) cm3 cm3 (P <0.05); the control group xenografts average volume (3.01 ± 0.76) cm3 Ad significantly less than the control group transplanted tumor volume (3.01 ± 0.76) 0.05)，见图1。">LacZ group xenografts average volume (2.75 ± 0.83) cm3 compared, the difference was not statistically significant (P> 0.05), as shown in Figure 1.
Comparison of Figure 1 xenograft tumor volume (cm3)
Fig 1 Comparison of volum among groups in
transplanted colorectal carcinoma (cm3)
2.2 transplantation tumor cell cycle distribution of PI and apoptosis rate comparison
Ad p27mt increased group transplanted tumor cells in G0/G1 phase, S phase and G2 / M phase cells decreased the, Ad of p27mt group and the other group difference was statistically significant (P <0.05), in the control group and the Ad LacZ group 0.05），见表1。">The difference was not statistically significant (P> 0.05), as shown in Table 1.
2.3 MMP 9 expression in the transplanted tumor
MMP 9 matrix area in the tumor area, coloring in the cytoplasm of cancer cells, brownish yellow granules, as shown in Figure 2. Expression of MMP 9 Ad p27mt group than the control group significantly decreased, as shown in Table 2.
a: control group; b: Ad p27mt group
Figure 2 MMP 9 expression in the transplanted tumor
Fig 2 Expression of MMP 9 in
transplanted colorectal carcinoma
p27 is a cell cycle negative regulatory factors, is a tumor suppressor gene . 0.05: AdLacZ group vs control group；* P<0.05: Adp27mt group vs AdLacZ group and control group抑制作用减弱，将导致细胞">Its abnormal expression (missing or decrease) of the cell cycle # P> 0.05: Ad LacZ group vs control group; * P <0.05: Ad p27mt group vs Ad LacZ group and control group inhibition weakened, leading to cell uncontrolled growth of cancerous . Ganoth  and Troncone  The results show that: p27 expression enhancements related to the role of the reduced ubiquitin mediated proteasome phosphorylation p27/187 bit of point threonine degradation pathway abnormalities. Therefore, this subject by intratumoral injection directly the 187 mutation p27mt recombinant adenovirus, study in the body (in vivo) inhibition of xenografts.
Park et al  found that inhibition of p27mt (mutant type p27) tumor cells than p27wt (wild type p27) stronger, so that the cell cycle arrest in G0/G1 phase, and a stronger pro-apoptotic effects. Jaen  further study find p27mt half-life of more than 12h, far more than the wild-type p27wt 2h. This topic by measuring tumor volumes the p27mt gene significantly inhibited the growth of colorectal cancer, the mechanism may be through the inhibition of cell proliferation and pro-apoptotic role. By the determination of the cell proliferation index and apoptosis rate detected p27mt can significantly inhibit cell proliferation, arrest cells in G0/G1 phase compared with the control group significantly induce apoptosis, apoptosis rate (37.9 ± 3.32)%.
The preliminary study of the subject found that colorectal cancer tissues showed low expression of p27, the lower the degree of differentiation, lower expression. p27 tumor differentiation and lymph node metastasis . In this study, by detecting the transplanted tumor tissue metalloproteinase 9 (MMP 9), to found p27mt reduce the expression of MMP 9. MMP 9 zymogen form in the cytoplasm, released into the extracellular physiological circumstances, the degradation of the extracellular matrix, and participate in the growth and development of the body and tissue repair a variety of physiological processes . MMP 9 gene disorder zymogen generate increased release into the extracellular degradation Ⅳ Ⅴ collagen, laminin, and other components, to undermine the integrity of the basement membrane, plays a very important role in the process of tumor invasion and metastasis . The observed here p27mt reduce the expression of MMP 9. The article also observed in the tumor growth within 28 days, not seen in tumor metastasis. p27mt inhibit tumor metastasis mechanisms or other mechanisms to reduce the expression of MMP 9, we will be further explored.
In summary, p27mt promote apoptosis through the inhibition of cell proliferation and inhibit the growth of colorectal cancer, and colorectal cancer metastasis certain extent.