1. Studies On Reversal Activity Of New Taxane Derivatives To Multi-drug Resistance And Its Mechanisms Of Action 2. Neurotrophic And Growth Factor Gene Expression Profiling Of Bone Marrow Stromal Cells Induced By Ischemic Brain Extracts And Their Effects O

Multidrug resistance (MDR) is a major impediment to successful cancer chemotherapy. MDR tumors show cross-resistance to multiple anticancer drugs of different chemical structure and mechanism of action, which, eventually, results in shortage of effective chemotherapy for patients. The complexity and multiplicity of MDR mechanism in tumors make overcome of MDR a great challenge. So far, the first, second and third generation drug resistance reversal agents have been developed over the past several years for overcoming tumor MDR. Unfortunately, clinical trials of the known MDR-reversal agents have been disappointing to date, for their limited reversal efficacy and severe cytotoxicity in patients. Therefore, more efforts are needed to be put into the search for novel, safe and potent reversal agents to meet the clinical requirement.Taxinine belongs to non-paclitaxel taxanes, with limited anticancer effects, but potent tumor MDR reversal activities. A series of novel taxane-based MDR reversal agents has been designed and synthesized from Sinenxan A by our institute. In our previous study, we obtained a leading compound, Sy1220, with potent reversing activity for tumor MDR in vitro and in vivo. The possible mechanisms by Sy1220 include inhibition of P-gp drug efflux function and the gene expression of multiple cell membrane transporters at mRNA and protein level. All leading compounds in this study are new taxane derivatives, with the hope that more powerful reversal agents could be discovered.In the first part of the dissertation, two taxinine leading compounds, coded as NBP14 and NBP36, were screened out from 42 chemicals. All of these 42 chemicals were designed with aims and synthesized from Sinenxan A. The further study focused on the effect of NBP14 and NBP36 in the anti-tumor activity and their reversing action of MDR in varieties of tumor MDR model cell lines. We also investigated the possible mechanisms of reversal of MDR by NBP14.Among the 42 taxinine derivatives, the better 15 compounds in terms of their MDR reversal activity in combination with paclitaxel against acquired MDR A549/Taxol cell line were chosen for further testing. After checking the activity of the 15 compounds to sensitize intrinsic MDR HCT-8 cell line to paclitaxel, two best compounds, NBP 14 and NBP36, were picked for the further study.The possible effects of NBP14 and NBP36 on cell growth inhibition were examined by SRB assay. The outcome showed that NBP14 and NBP36 had relatively weak cytotoxic activity, compared with paclitaxel. Their GI₅₀ values to various cancer cells typically were higher than 20μmol/L, and the results indicated that NBP14 and NBP36 had low toxicity to normal cells.Seven tumor cell lines were used to evaluate the reversal effects of NBP14 and NBP36, including one intrinsic MDR model, HCT-8 and 3 acquired MDR models with their anticancer drug sensitive parent cells, i. e. KB/V and KB, A549/Taxol and A549, and Be17402/5FU and Be17402. NBP14 and NBP36 exerted potent reversing activity for MDR when evaluated by cultured tumor cell lines. In intrinsic cell model, HCT-8, 5μmol/L NBP14 or 10μmol/L NBP36 could lead to a dramatic increase in paclitaxel-induced cytotoxicity without any appreciable cytotoxicity by itself. The reversal efficacy was higher than that of Verapamil at the same concentration. And in both KB/V and A549/Taxol cell lines, which were considered acquired MDR cell models, NBP14 and NBP36 could reverse the cytotoxicity of paclitaxel or vincristine, with the efficacy equal to or better than Verapamil at the same concentration. However, NBP14 and NBP36 failed to reverse MDR of Be17402/5FU and Be17402, so did Verapamil.The influence of NBP14 on apoptosis induced by paclitaxel in A549/Taxol cells was determined by AO/EB double stain, Hoechst 33258 stain and flow cytometry using DNA content analysis, which could also analyze the phase changes in cell cycle. Treatment with 10μmol/L NBP14 or 100nmol/L paclitaxel alone could not induce significant apoptosis in A549/Taxol cell lines; meanwhile, the combination of 10μmol/L NBP14 with 100μmol/L paclitaxel could significantly induce typical apoptosis characteristics in A549/Taxol cell lines in three different assays. FCM data showed that 10μmol/L NBP14 alone had no effect on cell cycle of A549/Taxol cell line, but combination of 10μmol/L NBP14 with 100nmol/L paclitaxel could significantly increased arrest at the G₂/M phase, which was the typical paclitaxel effect at high dosage in administration to cells. All the data from apoptosis assays showed that 10μmol/L NBP14 alone had week cytotoxic effect, but NBP14 could significantly enhance the apoptosis inducing action of paclitaxel, when combinations of drugs were administrated.Indirect immunofluorescent staining and tubulin assembly assay were used to evaluate the effects of NBP14 on the microtubulin, which usually presents the target of paclitaxel and vincristine. Results in immunofluorescent staining showed that NBP14 treatment alone had no effect on microtubulin system in the A549/Taxol cell line. However, coadministration to MDR with NBP14 tumor cells led to a dramatic enhancement in paclitaxel- or vincristine-induced anti-microtubulin effects. And in tubulin assembly assay, treatment in A549/Taxol cells with NBP 14 alone had no effect on polymerization of tubulin. When combined with paclitaxel or vincristine, NBP14 had no influence on their anti-tubulin efficacy, either. All the data indicated that the intact cells were indispensable for the enhancement of anti-tubulin effects of paclitaxel or vincristine by NBP14.Rhodamine123 accumulation assay was utilized to study the influence of NBP14 on the function of MDR1 and MRP1 transporters. Half-hour treatment with 5μmol/L and 10μmol/L NBP14, the intracellular rhodamine123 concentration increased 2.2-fold and 2.6-fold respectively, higher than the effect of 10μmol/L Verapamil in A549/Taxol cell line. And NBP14 did not influence rhodamine123 concentration in A549 cells.Over-expression of MDR1, MRP1, BCRP and LRP are common to MDR tumor cells. These membrane proteins act as ATP-dependent effiux pumps that extrude a wide variety of structurally unrelated compounds out of the cells and decrease their intracellular accumulation and efficacy, consequently, playing a major role in MDR. In this study, RT-PCR assay was conducted to detect the mRNA expression level of these 4 genes in varieties of MDR tumor cells. The results by RT-PCR assay indicated that the gene expression level of all 4 genes in HCT-8 cells was detectable, which may play the major role in its MDR. The levels ofMDR1, MRP1 and LRP in KB/V cells were significantly higher than those in KB cells. Compared with that in A549 cells, MDR1 and MRP1 mRNA expression level in A549/Taxol cells predominated. Furthermore, NBP14 regulation of MDR1 and MRP1 mRNA expression was examined in MDR A549/Taxol, human lung adenocarcinoma cell line by RT-PCR. 5μmol/L and 10μmol/L NBP14 could significantly decrease the expression level of MDR1 in MDR A549/Taxol cells. However, they had no effect on MRP1 level. The RT-PCR results showed that 20μmol/L NBP14 significantly inhibited both the transcription of MDR1 and MRP1 genes in MDR A549/Taxol cells.In conclusion, the possible mechanisms of MDR reversal of NBP14 in A549/Taxol cell line include inhibition of the function of P-gp and MRP1 drug efflux pumps and decrease of the transcription of MDR1 and MRP1. Both of these effects diminished the efflux of anti-cancer drugs from cells and therefore, enhanced the intracellular drug concentration. NBP14 and NBP36 are a new class of taxinine compounds with potent reversal activity for MDR with low cytotoxicity. Hopefully, results presented in this study will promote further development of these taxane derivatives as potential MDR reversal agents for clinical tumor therapy. Stroke is one of the leading causes of serious, long-term disability worldwide. Of all strokes, 87% is ischemic. Improved clinical outcome was seen only for patients with acute ischemic stroke when intravenous thrombolytic treatment was started within 3 hours. Regrettably, large amount of patients with ischemic stroke miss out on the optimum therapy. To expand the therapeutic window, more efforts have been taken to the alternative treatment, including cell transplantation, which has been greatly boosted by the development of stem cell research and application. Among several kinds of cell-based therapies, bone marrow stromal cells (BMSCs) are a strong therapeutic candidate. BMSCs enriched in vitro by self-renewal after isolation from adult bone marrow, virtually eliminate the ethical, immunological and logistical problems, associated with embryonic or adult neural stem cell therapies. Furthermore, increasing evidence shows that BMSCs survive, selectively migrate to injured areas and provide therapeutic benefits in a variety of CNS diseases, such as cerebral ischemia, traumatic brain injury, spinal cord injury and demyelinating disorders. These studies suggest the possibility of transplantation therapy using BMSCs for patients with various CNS disorders. However, the mechanisms by which BMSCs provide therapeutic benefits remain unclear.BMSCs, including stem and progenitor cells, are multipotent and capable of differentiation into mesodermal derivatives such as bone, cartilage, fatty tissue and even neural cells such as neurons. Although the transdifferentiation theory is attractive, it is inconsistent with in vivo data. Rodents after middle cerebral artery occlusion (MCAO) obtain therapeutic benefit within days, and very few BMSCs express neural markers. Clearly, weeks or months are needed for BMSCs to transdifferentiate into the lost neural cells and appropriately integrate into complex neural connections. Alternatively, orthotopic BMSCs naturally secrete a variety of cytokines and growth factors, which mainly support hematopoietic stem cells to differentiate into mature blood cells. Interestingly, the pattern and quantity of such functional secretion of BMSCs could be changed in response to their existing microenvironment. BMSCs in ischemic conditions increase the synthesis of some cytokines and growth factors.The first part of this study focused on the contribution of neurotrophic and growth factors produced by BMSCs to the therapeutic benefits. Rats were subjected to middle cerebral artery occlusion and the ischemic brain extract supernatant was collected to prepare the conditioned medium. The counterpart normal brain extract from non-ischemic rats was employed as the experimental control. Using microarray assay, we measured the changes of the neurotrophin associated gene expression profile in BMSCs cultured in different media. Furthermore, real-time RT-PCR and fluorescent immunocytochemistry were utilized to validate the gene changes. The morphology of BMSCs, cultured in the ischemic brain conditioned medium for 12 h, was dramatically altered from a polygonal and flat appearance to a fibroblast-like long and thin cell appearance, compared to those in the normal brain conditioned medium and the serum replacement medium. Forty-four neurotrophin associated genes in BMSCs were identified by microarray assay under all three culture media. Twelve out of the 44 genes, (7 neurotrophic and growth factor genes, and 5 receptor genes) increased in BMSCs cultured in the ischemic brain conditioned medium compared to the normal brain conditioned medium. Real time RT-PCR and immunocytochemistry validated that the ischemic brain conditioned medium significantly increased 6 out of 7 neurotrophic and growth factor genes, compared with the normal brain conditioned medium. These 6 genes consisted of fibroblast growth factor 2, insulin-like growth factor 1, vascular endothelial growth factor A, nerve growth factor beta, brain derived neurotrophic factor and epidermal growth factor.Astrocytes, as one of the main components of neurovascular units, could provide structural, trophic and metablic support for neurons. During post-ischemia, astrocytes are critical for neural and endothelial cell survival. Thus, in the second part of study, we investigated the effects of supernatant from bone marrow stromal cells cultured with brain ischemic tissue conditioned medium on mouse primary cultured astrocytic apoptosis and viability after hypoxia treatment. Our data indicate that ischemic brain extract conditioned medium could reduce cell death and apoptosis, and increase cell proliferation in astrocytes after hypoxia treatment. By performing real time RT-PCR, we observed that brain-derived Neurotrophic factor, basic fibroblast growth factor, glial cell line derived neurotrophic factor, and vascular endothelial growth factor gene expressions were enhanced by the ischemic brain extract conditioned medium. LY294002 inhibition assays showed that phosphoinositide3-kinase/threonine protein kinase pathway is involved in cell survival. Western blot analysis indicated that ischemic brain extract conditioned medium could activate this pathway and upregulate the phosphoration of Akt.All these findings suggest that small amount of implanted BMSCs may work as "small molecular factories" by secreting cytokines, neurotrophins, growth factors, and other supportive substances acutely after stroke, which further enhances large amount of cell survival and proliferation and decreases cell death post-ischemia. And these salvaged astrocytes could synthesize and secrete more neuroprotective and neurorestrorative factors, which could further activate the restorative properties on endogenous brain parenchymal cells. In summary, altered neurotrophic and growth factor gene expression in BMSCs, activated by ischemic brain extract conditioned medium, may be the initial spark for the therapeutic progress, which may produce therapeutic benefits in the ischemic brain via protection and activation of astrocytes.