Reverse Effects Of Diallyl Trisulfide On Drug Resistance Of MDR Human Osteosarcoma Cell Lines And Tumor Stem-like Cells

Background: Osteosarcoma is the most common primary bone malignancy in children and adolescents. Despite advances in surgery and multi-agent chemotherapy, long-term survival rates have stagnated at approximately 65%. The perplexing problem preventing successful chemotherapy is recurrence, metastasis and multidrug resistance of osteosarcoma. Although several drugs have been developed to treat osteosarcoma, the clinical effects are not so good. However, the hypothesis of tumor stem cells might point out the direction for future cancer research.The hypothesis of tumor stem cells have very important prospect in the study of mechanism tumorigenicity, metastasis and drug resistance. In fact, scientists had found the correlation of stem cell and cancer cells through observeing the similarity between cancer tissue and embryonic tissue. Recent reserches have shown that there are only a small fraction of tumor cells having unlimited proliferative capacity in leukemia and part of solid tumors cells. The small fraction of tumor cells, which are able to form new tumor foci, are referred to as tumor stem cells. The current concept of heterogeneity of tumor cells also includes the differences of tumor stem cells and non-tumor stem cells in addition to differences in response to chemotherapy and in the capacity of invasion and metastasis outside. Most tumor cells do not have unlimited capacity of proliferation except for tumor stem cells, which can generate a new self-renewing tumor stem cells and non-tumorigenic capacity of tumor cells.It is a big step forward in the process of exploring the mechanism of human tumor oncogenesis, etastasis and drug resistance mechanisms that tumor stem cell hypothesis. It has enriched the mechanism of human tumor oncogenesis in theory. In clinical practice, it remind us that the reason of our traditional failure chemotherapy is the tumor stem cell. The tumor stem cell over-express the protein of ABC transporters, which result in tumor stem cells escaping the current standard method of treatment, and to be the "source" of recurrence and metastasis. Therefore, the tumor stem cells have been considered a new target for the treatment of the tumor reserch, which has become a "radical" malignant hope.It is the research hotspot to seek for the tumor stem cells marker. In 1997, Bonnet reported that CD34+ CD38- cells of acute myeloid leukemia cells have the stem cell characteristics. Even though they accounted for only 0.2 percent of the total number of cells, but they are only oncogenous cells group in the SCID rat. In 2003, Singh SK found that CD133 + cells group, which are only a small number of the total cell number of the brain tumor cells, can form tumor cell balls in serum-free culture and CD133- cells group didn't. In the next year, they found that the CD133 + cells group of the brain tumor cells, which were transplanted into NOD / SCID mouse's brain, can cause brain tumors. But the CD133- cells group didn't. After 2006, more and more studies showed that CD133+ group cells is closely related to tumor stem cells. Suetsugua, Ricci-Vitiani L and O'Brien CA showed that they isolated CD133 + cells group from the hepatoma cell line Huh-7 and colon cancer tissues, respectively. And they proved that the cells group were tumor stem cells. At present, there is no recognized surface tumor stem cells markers of human osteosarcoma.Lot of the current studies suggest that tumor stem cells are the most fundamental reason that result in chemotherapeutic drugs resistant of tumor cells, which are the main obstacles to the treatment of malignant. The existing method of treatment of tumor is mainly directed against the majority of cells, rather than tumor stem cells. Tumor and normal stem cells have many similar characteristics, which are in the GO cell cycle phase and expression of specific ATP-binding cassette (ATP-binding cassette, ABC), such as ABCB1, ABCG2 and so on. These membrane pump-mediated resistance elements resulted in their natural resistance to chemotherapy. ABCB1, ABCG2 from tumor tissues are tumor multidrug resistance gene. ABCB1 encode P-gp, and ABCG2 encode BCRP. These transporter protein, which use the energy generated for ATP decomposition, will pump the intracellular drugs out and protect tumor stem cells from cytotoxic drug damage. Because tumor stem cells over expressed ATP-binding cassette, chemotherapeutic drugs can be pumped out of cells and intracellular drug concentrations be reduced. After tumor chemotherapy, there are still a small number of tumor stem cells survive. We believed that this is an important mechanism of the tumor drug resistant, recurrence and metastasis.In order to look for the way of reversal multidrug resistance of tumor, scientists have experimented with many drugs, such as calcium channel blockers, immunosuppressive agents, as well as hormone drugs and so on. They have achieved some results. However, some side effects of these drugs have limited the clinical application. Therefore, many researchers began to study some natural medicines. The ingredients of garlic, DAS, has been proved to have the ability to reverse P-gp-mediated leukemia K562 multidrug resistance at the low toxicity doses. In recent years, epidemiological studies have confirmed that garlic has a close relationship with the many types of human cancer incidence rates drop. This should be attributed to crush fresh garlic from the extracted organic sulfur compounds (organosulfur compounds, OSCs), mainly including the DAS (diallyl sulfide, diallyl sulfide 1), DADS (diallyl disulfide, diallyl disulfide ) and DATS (diallyl trisulfide, diallyl trisulfide, also known as allicin). We ever studied the role of DATS on osteosarcoma cell line Saos-2. We found that DATS is able to inhibite osteosarcoma cells. However, as for the question of the DAS and DATS have a similar reversal of P-gp-mediated multidrug resistance role, there are not related studies yet.In this study, we studied the tumor stem cell markers CD133 expression on human osteosarcoma cells. And we sort out the CD133-positive (CD133 +) group of cells from Saos-2 osteosarcoma cell lines using magnetic separation methods. We studied these cells differentiation, cell cycle, tumorigenicity and MDR1, Sox2 gene expression, which are related to multidrug resistance-associated and stem cell regulation and control. Additionally, we set up multi-drug resistance in osteosarcoma line Saos-2/R using methotrexate (MTX)-induced concentration ways. The ability of DATS reversing drug-resistant osteosarcoma cells were assessed. Finally, the imact of MTX on CD133 marker percentage were studied after DATS reversing drug-resistant of Saos-2/R. Objective: To analyze the authentication osteosarcoma tumor stem cell surface molecular markers. To set up drug-resistant osteosarcoma cell lines and research DATS's potential of reversing drug resistance in osteosarcoma cell.To study the imact of MTX on CD133 marker percentage before and after DATS reversing drug resistance of osteosarcoma tumor cells.Methods: Immunohistochemical study of CD133 expression: 55 cases osteosarcoma conventional paraffin-embedded tissues specimens come from Pathology Department Qilu Hospital Shandong University, including 4 cases parosteal osteosarcoma, 13 cases fibroblast-type osteosarcoma , 12 cases chondroblastic osteosarcoma and 26 cases Osteoblastoma osteosarcoma. They were cut into 3μm standby. Check logarithmic growth phase Saos-2 cells subcultured in Petri dish with the coverslip, which has been sterilized. When the cells have adhered to coverslip after being cultured for 24h, we use 70% ethanol to fix the cells for 20 minutes standby. CD133 expression by flow cytometry analysis: To analyze percentage of CD133 expression in the logarithmic phase of osteosarcoma cell line Saos-2 cells. After removing dead cells using Dead Die cell separation kit, cells were incubated with monoclonal anti-CD 133 conjugated to PE and detected the percentage of CD133 expression using a FACScan. CD133-positive Saos-2 cell differentiation: To detect the changes of CD133 positive percentage and the cell cycle before and after CD133-positive Saos-2 cell incubated in complete medium and. To remove dead cells using MACS MicroBeads separation, then sort out CD133 + cells from Saos-2 cells and analyze changes of CD133 positive percentage at 0^th, 3^th and 10^th day after incubating in complete culture medium. And to analyze changes of the cell cycle at 0^th and 10^th day after incubating in complete culture medium. MDR1, Sox2 gene expression of CD133 + / - cells, by Real-time PCR analysis: Using Real-time PCR to analyze MDR1 and Sox2 expression of CD133+ cells, which are related to multidrug resistance and stem cell differentiation regulation. To remove dead cells using MACS MicroBeads separation, then sort out CD133 +/- cells from Saos-2 cells. According to operation Manual, RNA extraction and reverse transcription-line were done and then MDR1 and Sox2 expression were analyzed by Real-time PCR. CD133 + / - cells colony forming test: To analyze the differences of CD133+/- cells tumorigenicity using flat cell cloning test. Using MACS to remove dead cells and sort out CD133+/-cells from Saos-2 cells according to operating manuals. To dilute make cell suspension according to 50,100 and 200 cells per dish density gradient. They were inoculated in the Petri dish with 10ml pre-heating at 37℃culture medium. To shake up the Petri dish so that cells dispersed sloshing uniform. And then the Petri dish were plate into the incubator at 37℃, 5% CO2 and saturated humidity environment. After the static cultivate for three weeks, the Petri dish were placed on the plate under the microscope and the number of clones were counted directly by the naked eye. Then, Cell s colony forming efficiency was calculated according to the formula: cells colony forming efficiency (%) = Cloning number / inoculation cell number×100. A cell line resistant to MTX cytotoxicity (named for the Saos-2 / R) was established in our laboratory by progressive adaptation of MTX-sensitive Saos-2 parental cells to increasing concentrations of MTX. To study the P-gp (MDR1) expression of Saos-2/R using RT-PCR and Western blotting. To detect changes in drug sensitivity on a variety of chemotherapy drugs and to determine whether they have multi-drug resistance with MTT test. DATS reverse drug resistance research: Saos-2 / R cells were incubated for 48h with the complete medium containing the gradient concentration of DATS of incubated with 48h. To detect the changes of P-gp (MDR1) expression using RT-PCR and Western blotting method to analyze the lowest DATS reversing concentration. Assay of MTX reducing the tumor stem-like cells after DATS reversing drug resistance of Saos-2/R: Saos-2 / R cells were incubated with complete medium containing DATS of lowest reversing concentration, and the MTX gradient concentration for 48h. And the Saos-2 / R incubated in complet medium without DATS to be the control cells. They also contain the same MTX concentration gradient. To analyze the changes of CD133+ cells percentage using flow cytometry analysis.Results: CD133 + cells were is commonly found in human osteosarcoma tissue accounting for about 5-14%, while they were also found in the human osteosarcoma cell line Saos-2 accounting for around 5%. CD133 + cells were put in complete medium after being sorted out from Saos-2 cells using MACS. And then, the percentage of CD133 + cells were detected by FCM. We found the percentage declined rapidly with the extension of time to cultivate. At the same time, the cell cycle at 10^th day is similar to nomal Saos-2 cell cycle, and there is significant difference compared with that at 0^th day. We also found that CD133 + cells have higher rates of colony-forming cells than that of CD133- cells. And CD133 + cells over-expressed MDR1 and Sox2 genes compared with CD133- cells. As mentioned earlier, it is an important reason of tumor stem cells MDR that MDR1 over-expression. And Sox2 gene is one of important core member of stem cell gene regulatory network. In the light of the above, we believe that CD133 + cells group of Saos-2 cells have some of key features of tumor stem cells. We call them tumor stem-like cells. We use CD133 as a marker to study the percentage of changes of CD133+ Saos-2/R cells after the Saos-2 / R drug-resistant being reversed by DATS. We assume that groups of CD133 + cells percentage will decline after drug resistance being reversed by DATS for not tolerating the toxicity of chemotherapy drugs. We compared the CD133 percentage changes of Saos-2/R incubated in complete medium with MTX concentration gradient with/without 10uM DATS. We found that the presence of CD133 + cells is stable in the group absence of DATS, while the percentage declined gradiently in the presence of 10μM DATS.Conclusion: CD133 + cells were is commonly found in human osteosarcoma tissue and human osteosarcoma cell line(Saos-2) with a small amount. The CD133 + group of Saos-2 cells are at a quiescent cell cycle period and over-express key regulatory genes of nomal stem cells and drug resistance-related genes. And they have highly tumorigenic characteristics. These results showed CD133 + group of Saos-2 cells have the characteristics of tumor stem cells. We called tumor stem-like cells. The drug-resistance cells line Saos-2/R, which we set up using MTX inducing, have MDR characteristics. DATS can effectively reduce P-gp (MDR1) expression of Saos-2/R and reverse its drug resistance. After the Saos-2 / R drug-resistant being reversed, CD133 + cells percentage of Saos-2/R reduce for not being tolerated the toxicity of MTX. DATS may reverse the role of drug resistance of tumor stem-like cells while reversing drug resistance of Saos-2/R.