Effects And Mechanism Of Berberine On The Treatment Of Osteosarcoma

Osteosarcoma is a primary malignant tumour of the skeleton characterised by the direct formation of immature bone or osteoid tissue by the tumour cells. The tumour has a slight predilection for males, with a reported male-to-female ratio of around 1.5:1 to 2:1. In the long bones, osteosarcoma usually originate in the metaphysis, it was most common in the distal of femur and the proximal of tibia. Micro-metastases were existed in (80-90)% of patients at onset, so osteosarcoma is a systemic disease with micro-metastasis. Therapy option for osteosarcoma was a comprehensive treatment based on the surgery. Before the advent of chemotherapy the prognosis of osteosarcoma was poor, with 5-year disease-free survival rates ranging between 15% and 20% after surgical treatment. Chemotherapy has changed the survival rate range to 75% to 80%. The likelihood of local recurrence is 5% to 7%, and is related to surgical resection margins and responsiveness to chemotherapy.Therapeutic researchs of osteosarcoma were focused on screening the sensitive treatment methods and drugs. Other treatment options or drugs should be considered in patients with drug-resistantance or poor response to chemotherapy drugs (usually tumor recurrence or metastasis is more likely); another research focus is to find new treatment targets in order to improve the poor response of tissue to chemotherapeutic drugs, enhance the role of chemotherapy, reduce the toxicity of chemotherapy drugs and reduce the side effects of chemotherapy; another research is looking for new anticancer drugs or old drugs with new anti-tumor activity(belonged to development of new drugs). Berberine, also known as huangliansu, was a common isoquinoline alkaloids, C20H18NO4.CL was its molecular formula. It exists in many plants in the genus of Berberidaceae. Many studies have evidence that berberine hydrochloride (commonly known as berberine) could inhibit the activities of Shigella dysenteriae, Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus, Salmonella typhi and amoeba, has been widely used in treatment to intestinal infections and dysentery; recent years, studies have found that the BR was cytotoxic and it can inhibit the activities of tumor cells such as liver cancer, leukemia cell and other malignancies cells by inhibiting protein synthesis in cancer cells, blocking cell cycle, promoting apoptosis; berberine hydrochloride have synergistic effect with hydrochloric acid cytarabine in vitro. Another study also reported that berberine hydrochloride can induce the G1 arrest and apoptosis depended on the p53 gene in U2OS cells. In this study, effects of berberine on the proliferation, apoptosis, invasion, metastasis and the possible molecular mechanisms in osteosarcoma cell line MG-63, U2OS were studied in vitro studies; and the therapeutic effect and possible molecular mechanism were further studied in subcutaneous transplantation model of osteosarcoma with MG-63 cell in nude mice. Effects of berberine on apoptosis and invasion and metastasis of MG-63, U2os cell and its possible molecular mechanism were studied at the whole animal level and the cell level.In Vitro Study Effects and Mechanisms of BBR on Osteosarcoma Cells Proliferation and ApoptosisResearch aims:The effects and mechanisms of BBR on osteosarcoma cells proliferation and apoptosis were investigated.Research Methods:1. MG-63 and U2OS cells were respectively cultured in McCoy's 5a Medium Modified and DMEM media containing 10% FBS, and were kept in an incubator with 37℃and 5% CO2. When grow to 90% confluence in 75 cm2 flasks, MG-63 and U2OS cells were passaged by 0.25% trypsin, the appropriate intervention were given according to the experimental need.2. After the first step in processing, the cell count kit-8 was carried out to measure the effects of BBR on MG-63 and U2OS cells proliferation;3. After the first step in processing, flow cytometry assays were carried out to determine the effects of BBR on MG-63 and U2OS cells apoptosis;4. After the first step in processing, p38MAPK and JNK inhibitors SB203580 and SP600125 and MG63 and U2OS cells, as well as BBR were incubated together for 24 h, and then flow cytometry assays were carried out to determine the effects of BBR on MG-63 and U2OS cells apoptosis;5. After the first step in processing, MG-63 and U2OS cells were randomly divided into Control group, Vehicle group, BBR 10 ug/ml group, BBR 50 ug/ml group and BBR 100 ug/ml group, and then stimulated with different varieties and different concentrations of BBR. After 24 h, the caspase-3 activity was then determined by caspase-3 colorimetric assay kit.6. After treated with BBR for 24 h, MG-63 and U2OS cells were respectively collected, and proteins were extracted. Western blot was carried out to detect Bcl-2 and Bax expression in MG-63 and U2OS cells cells.Research results:1. BBR significantly promoted MG-63 and U2OS cells proliferation in a time- and dose-dependent manner.2. BBR significantly induced MG-63 and U2OS cells apoptosis. P38MAPK inhibitor SB203580, JNK inhibitor SP600125 significantly inhibited MG-63 and U2OS cells apoptosis induced by BBR. The results showed that BBR significantly increased the ratio of apoptotic cells. The ratios of apoptotic cells for MG-63 cell in the group of Control, DMSO and BBR 10 ug/ml group, BBR 50 ug/ml group and BBR 100 ug/ml group were (3.570±0.661)%, (5.820±0.456)%, (7.930±0.511)%, (13.630±1.352)% and (22.187±1.962)% respectively; the ratios of apoptotic cells for U2OS cell in the group of Control, DMSO and BBR 10 ug/ml group, BBR 50 ug/ml group and BBR 100 ug/ml group were (4.083±1.301)%, (7.803±0.626)%, (10.630±0.534)%, (17.470±1.984)% and (27.660±2.088)% respectively. The differences of the ratio of apoptotic cells for MG-63 and U2OS cells between group of BBR 10 ug/ml, BBR 50 ug/ml and BBR 100 ug/ml and DMSO group were statistically significant (P<0.05).3. The activity of caspase-3 in MG-63 and U2OS cells started to increase after 8 h interfered with 100 ug/ml BBR and the caspase-3 activity was significant higher than that in the Vehicle group at 12 h, the activity decreased at 24 h. The results showed that, the activity unit in MG-63 cell in the group of Control were 6.68±0.371,7.051±1.575 and 6.574±0.822 respectively; the group of DMSO were 7.423±1.113,14.103±0.525 and 10.577±0.787 respectively; at the different time in the group of BBR 100 ug/ml were 16.857±1.174,21.795±0.381 and 8.97±0.076; the activity unit in U2OS cell in the group of Control were 7.550±1.086,6.680±1.113 and 8.289±0.934 respectively; the group of DMSO were10.903±0.236,10.886±3.156 and 11.752±0.773 respectively; at the different time in the group of BBR 100 ug/ml were 15.464±1.754,21.129±1.138 and 9.135±0.243.4. The results showed that BBR significantly decreased the expression levels of Bcl-2 (P<0.05) in MG-63 and U2OS cells, and the expression levels of Bax in MG-63 and U2OS cells were increased (P<0.05).In Vitro Study Effects and Mechanisms of BBR on Osteosarcoma Cells Migration and InvasionResearch aims:The effects and mechanisms of BBR on osteosarcoma cells migration and invasion were investigated.Research Methods:1. MG-63 and U2OS cells were respectively cultured in McCoy's 5a Medium Modified and DMEM media containing 10% FBS, and were kept in an incubator with 37℃and 5% CO2. When grow to 90% confluence in 75 cm2 flasks, MG63 and U2OS cells were passaged by 0.25% trypsin.2. After the first step in processing, the wound healing test was carried out to measure the effects of BBR on MG-63 and U2OS cells ability of migration;3. After the first step in processing, the transwell invasion test was carried out to determine the effectsof BBR on MG-63 and U2OS cells invasive ability;4. After treated with BBR for 24 h, MG-63 and U2OS cells were respectively collected, and proteins were extracted. Western blot was carried out to detect MMP-2 and MMP-9 expression in MG-63 and U2OS cells cells.Research results:1. BBR significantly decreased MG-63 and U2OS cells ability of migration. The results showed that the ratios of migration in MG63 cell in the group of Control, DMSO and BBR 10 ug/ml group, BBR 50 ug/ml group and BBR 100 ug/ml group were (66.3±7.1)%, (63.7±5.7)%, (53.3±3.1)%, (41.3±4.0)% and (26.3±3.2)%; the ratios of migration in U2OS cell in the group of Control, DMSO and BBR 10 ug/ml group, BBR 50 ug/ml group and BBR 100 ug/ml group were (72.7±7.5)%, (68.3±7.6)%, (51.3±3.2)%, (35.0±5.0)% and (21.7±10.4)%. The differences of the ratio of of migration in MG-63 and U2OS cells between group of BBR 10 ug/ml, BBR 50 ug/ml and BBR 100 ug/ml and DMSO group were statistically significant (P<0.05); The differences of the ratio of of migration in MG-63 and U2OS cells between group of DMSO and Control group were no statistically significant (P>0.05).2. BBR significantly decreased MG-63 and U2OS cells ability of invasion. The results showed that the cell number cross through the membrane in MG-63 cell in the group of Control, DMSO and BBR 10 ug/ml group, BBR 50 ug/ml group and BBR 100 ug/ml group were 852.7±53,835.7±33.1,681±22,514±31.8 and 331±32.9; the ratios of migration in U2OS cell in the group of Control, DMSO and BBR 10 ug/ml group, BBR 50 ug/ml group and BBR 100 ug/ml group were 974.0±60.0,950.0±16.0,715.0±95.0,627.3±23.2 and 447.3±11.9. The differences of the cell number cross through the membrane in MG-63 and U2OS cells between group of BBR 10 ug/ml, BBR 50 ug/ml and BBR 100 ug/ml and DMSO group were statistically significant (P<0.05); The differences of the cell number cross through the membrane in MG-63 and U2OS cells between group of DMSO and Control group were no statistically significant (P>0.05).3. The results showed that BBR significantly decreased the expression levels of MMP-2 andMMP-9 in MG-63 and U2OS cells (P<0.05).In Vivo Study Effects and Mechanisms of BBR on Growth and Invasion of Subcutaneous Transplantation Model of Osteosarcoma with MG-63 Cell in Nude MiceResearch aims:The effects and possible molecular mechanisms of BBR on growth and invasion of subcutaneous transplantation model of osteosarcoma with MG-63 cell in nude mice were investigated.Research methods:1. MG-63 cells were cultured in DMEM media containing 10% FBS, and were kept in an incubator with 37℃and 5%CO2. When grow to 90% confluence in 75 cm2 flasks, MG63 cells were passaged by 0.25% trypsin.2.30 4-week-old healthy male BALB/C-nu-nu mice were tagged and weighed, and every three animals were feed in one cage in SPF standard animal holding rooms at the Experimental Animal Center of Tongji Medical College Huazhong University of Science and Technology. The room temperature was 22℃～26℃, the relative humidity was 50%～80% and day light for 12 h in the animal holding rooms, standard chow diet and water were given freely. All experimental animals operating procedures are in line with the NIH laboratory animal care and usage guidelines, as well as the standards set by the Chinese Academy of experimental animal management practices. 3. Animals were subjected to a one-week adaptation period. The MG-63 cells were digested by 0.25% trypsin, washed by 1×PBS, resuspended in normal saline.1×107 MG-63cells were injected into the subcutaneous of the armpit of nude mice. The transplanted tumor was removed in a sterile operating room, when the transplanted tumor grows to about 1.5 cm×1.5 cm sizes. The transplanted tumor was ground into a cell suspension adjusted to the concentration of 1×107/ml.1×107/ml transplanted tumor cells were injected into the subcutaneous of the armpit for all the nude mice, then the subcutaneous transplantation model of osteosarcoma with MG-63 cell in nude mice were established.4. The subcutaneous transplantation models of osteosarcoma with MG-63 cell in nude mice were randomly divided into three groups (10 mice for each group):the negative control group (sterile saline were given), the positive control group (intraperitoneal injection of doxorubicin 1 ug/g. d) and the BBR group (BBR 50 ug/g. d were given). The above interventions were carried out after the subcutaneous tumor appeared. The volumes of subcutaneous tumor were measured with a vernier caliper every other day until the end of the experiment. The subcutaneous tumor were removed and weighed, at the end of the experiment. Part of the tumor tissue were taken and placed in liquid nitrogen, and then transfered into the -80℃refrigerator, and some of the above tissues were fixed with formaldehyde solution and stored at room temperature;5. The subcutaneous tumor experiment was carried out to evaluate the effects of BBR on tumor proliferation in vivo.6. The immunohistochemistry was carried out to measure the expression of MMP-2, MMP-9 and nm-23 in subcutaneous transplanted tumors.Research results:1. MG-63 cells were transplanted into the subcutaneous of the armpit of nude mice, in accordance with the above method. The subcutaneous tumor emerged at the 12th day after planting. The subcutaneous tumor were removed and weighed, and the volumes of subcutaneous tumor were measured at the end of the experiment. The results show that the volume of subcutaneous tumor in the negative control group was (5.4016±0.3774) cm3 in the 36th day; the volume of subcutaneous tumor in the positive control group was (3.8484±.1963) cm3; the volume of subcutaneous tumor in the BBR group was (4.2525±.2822) cm3; The weight of subcutaneous tumor in negative control group was (2.9471±1.3682) g; the weight of subcutaneous tumor in positive control group was (1.285±0.1864) g; the weight of subcutaneous tumor in BBR group was (1.8452±.6963) g. There were significant difference in the weight and volume of the subcutaneous tumor in the positive control group and the BBR group, compared with that in the negative control group (P<0.05); there were no significant difference in the weight and volume of the subcutaneous tumor in the BBR group, compared with that in the positive e control group (P>0.05).2. At the end of the experiment, part of the tumor tissue were taken and fixed with formaldehyde solution. The expression of MMP-2,MMP-9 and nm-23 in the tumor tissue were detected by immunohistochemical method. The results showed that there was significant difference in the expression of MMP-2 and MMP-9 in the positive control group and the BBR group, compared with that in the negative control group (P<0.05); there was no significant difference in the expression of MMP-2 in the BBR group, compared with that in the positive control group (P>0.05); there was significant difference in the expression of MMP-9 in the BBR group, compared with that in the positive control group (P<0.05). There was significant difference in the expression of nm-23 in the positive control group and the BBR group, compared with that in the negative control group (P<0.05); but, there was no significant difference in the expression of nm-23 in the BBR group, compared with that in the positive control group (P>0.05).Statistical AnalysisContinuous data were expressed as means±S.D. Comparisons between groups were performed by a one-way analysis of variance. Two-way analysis of variance was used to examine differences in response to treatments and between groups, with post hoc analyses performed using the Student-Newman-Keuls method. Statistical significance was defined as P<0.05.Conclusions1. BBR significantly promoted MG63 and U2OS cells proliferation in a time- and dose-dependent manner.2. BBR induced MG63 and U2OS cells apoptosis may via activation of p38MAPK and JNK signaling pathways.3. BBR significantly decreased MG63 and U2OS cells ability of invasion may be achieved by down-regulating the expression of MMP-2 and MMP-9.4. The subcutaneous transplantation model of osteosarcoma with MG63 cell in nude mice was established successfully.5. BBR can inhibit the proliferative capacity of the subcutaneous transplantation model of osteosarcoma in nude mice in some extent.6. BBR can improve the cachexia state of the subcutaneous transplantation model of osteosarcoma in nude mice.7. BBR can reduce the capacity of invasion and metastasis in the subcutaneous transplantation model of osteosarcoma in nude mice, the possible molecular mechanism was the up-regulated expression of nm-23 and the down-regulated expression of MMP-2 and MMP-9.