In Vitro And In Vivo Effects Of HBMP2 And 9 On Human Osteosarcoma

Osteosarcoma is the most common malignant tumor primarily occurred in bone with characteristics of high malignancy, low five-year survival rate and predilection in children and teenagers. It's difficult to be early diagnosed and usually has metastasized to distant places when discovered. Although the current therapeutic regimen, the neoadjuvant chemotherapy combined with limp salvage surgery, is better improved than before, 25%~30% patients still suffered recurrences which threaten their lives. Therefore, it is very important to early diagnose and control recurrence and metastasis effectively.Bone morphogenetic proteins (BMPs), members of transforming growth factorβ(TGFβ), were first named for induction of bone and cartilage formation. But more and more researches found that BMPs had effects on formation and development of many systems or organs besides induction of bone formation. For example, BMP2 plays a role in heart morphogenesis(Callis et al., 2005) and in neural stem cells (White et al., 2001), BMP7 in kidney formation (Simic and Vukicevic, 2005) and diverse BMPs have unique roles in reproductive organs (Shimasaki et al., 2004; Tsumaki and Yoshikawa, 2005). And recent studies found that BMP2 could enhance the motility of prostate cancer cells and BMP4 inhibits proliferation and induces apoptosis of multiple myeloma cells. BMPs have already been reported to be related with lung cancer, gastric carcinoma, breast cancer, prostatic carcinoma, neuroglioma, osteosarcoma et al. At present, there are many reports about different effects of BMPs on osteosarcomas.In our study, BMP2 and BMP9 genes were mediated by adenovirus and transfered into human osteosarcoma cell lines MG63, U2OS and 143B; Then in vitro, cell proliferation, apoptosis, migration were studied andβ-catenin-the key molecule of Wnt/β-catenin signal was detected; In vivo, effects of hBMP2 and 9 on subcutaneous transplantation tumor model of athymic nude mice constructed with 143B cell lines were studied. Total program was divided into three parts, design, protocols and results were as follows:1. Immunocytochemistry(ICC)and Western blot were used to detect the expression of hBMP2 and 9 in MG63,U2OS and 143B cell lines:1.1 Endogenous expression of hBMP2 and 9 in MG63,U2OS and 143B cell lines:We found there were expression of BMP2 and 9 in three cell lines by two methods, but the expression levels were different. ICC showed BMP2 and 9 were mainly located in cytoplasma.1.2 After infected by Adenovirous-BMP2 (AdBMP2) or Adenovirous-BMP9 (AdBMP9) for 72h, 80%～90 % cells of MG63,U2OS and 143B expressed green fluorescent protein(GFP),which indicated adenovirous infection was efficient. And ICC showed expression of corresponding BMP was increased in cytoplasma and emerged in nucleus.The IOD value of hBMP2 in MG63, U2OS and 143B after infected by AdBMP2 analyzed by Image Pro Plus software were 1.27, 2.64 and 1.98 times of those in AdGFP group. Western blot showed a stronger reaction band after infection. Analyzed by Quantity-one 4.31 software, the gray value of BMP2 in AdBMP2 groups of MG63, U2OS and 143B were 1.41, 1.97 and 1.34 times of those in AdGFP group. It indicated that the genes of BMP2 and BMP9 could express in the target cells transfected by recombinated adenovirous-AdBMP2 and AdBMP9.2. In vitro experiments:2.1 Experiment groups were as follows :①Blank control:MG63,U2OS and 143B cell lines without any treatments;②experimental control:osteosarcoma cell lines infected by Adenovirous-GFP(AdGFP);③experiment group: cells infected by AdBMP2 and 9 respectively. 2.2 Protocols:Cell proliferation, apoptosis and migration of every groups of MG63, U2OS and 143B cell lines were detected by MTT Cell Proliferation Assay and Typan blue exclusion Assay, Hoechst/PI double-stained, and wound healing assay, respectively.β-catenin levels of every groups were investigated by ICC and Western blot.2.3 Results:1) Effects of hBMP2 and 9 on proliferation of human osteosarcoma cell lines MG63, U2OS and 143B ①MTT assay:After infected by AdBMP2 and 9,the absorbances at 570nm ( A570 ) of all experiment groups were decreased in a time-dependent manner in 72h, while A570 of two control groups were time-dependently increased. The differences between experiment groups and controls were significant(P<0.01), while the differences between every blank control and corresponding experimental control were not significant(P>0.05).②Typan blue exclusion Assay:The results were consistent with MTT assay.2)Effects of hBMP2 and 9 on apoptosis of human osteosarcoma cell lines MG63, U2OS and 143BCells of every group were viewed under fluorescent microscope after double-stained by Hoechst/PI and apoptosis cells were counted. There were apoptosis cells in all AdBMP2 and 9 experimental groups. The nuclei of apoptosis cells were stained to strong blue, the shapes were changed from normal ellipse to nicked, even like crescent, dumb bell along with prolong of time. Chromatin of some cells were highly condensed and located in the verges of nuclei. There also were apoptosis cells in every blank control group and corresponding experimental control group, but they were much fewer and in an earlier stage—their nuclei nicked a little.Count apoptosis cells and calculate the apoptosis rates: the apoptosis rates of every experiment group time-dependently increased and were significantly different with corresponding experimental control group(p<0.01)while without significant difference between blank control and experimental control.For example, at 24h, 48h and 72h, the apoptosis rates of 143B in blank control, AdGFP group, AdBMP2 group and AdBMP9 group were 11.5%, 22.5% and 32%; 12.5%, 26% and 32%; 20.5%, 46% and 74.5%; 18.5%, 54.5% and 69.5%, respectively. The apoptosis rates of AdBMP2 group and AdBMP9 group were 1.64, 2.49 and 2.87 times; 1.48 times, 2.95 times and 2.67 times of AdGFP group respectively. It indicated that hBMP2 and 9 could induce apoptosis of human osteosarcoma cell lines MG63, U2OS and 143B.3)Effects of hBMP2 and 9 on migration of human osteosarcoma cell lines MG63, U2OS and 143BWound healing assay:①After scarification for 24h,the wounds of MG63 blank control and AdGFP-MG63 were all healed while those of AdBMP2-MG63 group and AdBMP9-MG63 group were only healed 47.5% and 34.1%.②After scarification for 72h,the wounds of U2OS blank control and AdGFP- U2OS were all healed while those of AdBMP2- U2OS group and AdBMP9- U2OS group were still healed 23.7% and 43.1%.③The wound healing speeds of AdBMP2-143B and AdBMP9-143B were obviously lower than those of corresponding control. While wounds of two control groups were completely healed after 20h.④Among the three cell lines, migration of 143B was the fastest, MG63 ranked the secondary and U2OS ranked the third.4)Effects of hBMP2 and 9 on the level ofβ-catenin--the key signal molecule of Wnt/β-catenin in human osteosarcoma cell lines MG63, U2OS and 143BIn MG63: ICC showed thatβ-catenin of blank control and AdGFP control in MG63 were strongly positive,which appeared lots of buffy particles in cytoplasma and a few in nuclei. Andβ-catenin of AdBMP2 group and AdBMP9 group were positive. And the IOD values of AdGFP group was significantly higher than both AdBMP2 group and AdBMP9 group analyzed by Image Plus Pro(p<0.01)while IOD values of two control were not obviously different(p>0.05).The results of ICC in U2OS and 143B were similar to that of MG63. It indicated that AdBMP2 and 9 could down-regulate theβ-catenin level of MG63, U2OS and 143B.3 In vivo experiments:3.1 Protocols:143B cells were infected by AdBMP2 and 9 in experiment groups and by AdGFP in experimental control group. No treatment in blank control. Then the cells of each group were subcutaneously injected into athymic nude mice and processes of tumor growth were documented and compared to investigate the effects of hBMP2 and 9 on human osteosarcomas.3.2 Results:After injection for 60 days, the mean tumor volumes of blank control, AdGFP group, AdBMP2 group and AdBMP9 group were 3141±1488mm3, 3475±1544mm3, 1116±1200mm3 and 582±400mm3, respectively. There were no significant difference between the blank and the AdGFP group(p>0.05) while there were significant difference between AdGFP group and experiment group(sp<0.01). According to the tumor growth curve, tumors grew quickly in control groups while slow in experiment groups.It indicated that BMP2 and BMP9 could inhibit human osteosarcoma in vivo, which was consistent to that BMP2 andBMP 9 could inhibit cell proliferation of osteosarcoma cells in vitro.To sum up, the endogenous expression of hBMP2 and 9 in osteosarcoma cells MG63, U2OS and 143B and in vitro effects of hBMP2 and 9 on cell proliferation, apoptosis and migration on the three cell lines andβ-catenin level and Subcutaneous transplantation tumor model of athymic nude mice in vivo effects of BMP2 and BMP9 were investigated, the main conclusions are as follows:1. There were endogenous expression of hBMP2 and 9 in osteosarcoma cells MG63, U2OS and 143B.2. In vitro, exogenous hBMP2 and 9 could inhibit cell proliferation and migration of MG63, U2OS and 143B and induce their apoptosis, at the same time, down-regulate theβ-catenin level. It indicated that hBMP2 and 9 could inhibit the biological activities of MG63, U2OS and 143B maybe by Wnt/β-catenin signal.3. Subcutaneous transplantation tumor model of athymic nude mice constructed with 143B cell lines had the characteristic of quick growth and long survival time of 2 months. So it is a nice animal model for osteosarcoma research.4. In vivo, exogenous hBMP2 and 9 could obviously inhibit the growth of osteosarcoma.In a word,this study relatively systematically investigated the in vitro and in vivo effects of exogenous hBMP2 and 9 on osteosarcoma and provided evidences for elucidation of biological effects, proper mechanism of BMPs on osteosarcoma and provide clues for searching new target for osteosarcoma therapy.