| ObjectiveBrain glioma is a kind of common malignant diseases in human nerve system , chemotherapy after surgery is a very important method to treat the disease. Due to blood - tumor barriers (BTB) , it's difficult to deliver large - molecule anticancer drugs to tumor sites, which often leads to poor effects of chemotherapy. In order to accurately delivery drugs to tumors and relieve the side effects, we need a strategy to selectively open the BBB of tumors. Recently, Bradykinin was shown transiently increasing the permeability of the BTB without normal brain involvement. Bradykinin's analog, Cereport (also called RMP - 7) was exploited to Phase â… ,â…¡ clinical studies. However, when the patients with glioma were injected RMP - 7 combined with carboplatin, the Ki values, which represented the permeability of BBB, were variable among patients, which might leads to unreliable curative effects after RMP - 7 administration. As a result, RMP -7 is not been permitted in clinical therapy.We suppose that the possible reasons for this phenomenon are close related with the mechanism of BK - mediated BTB permeability increase, which remains unclear. It might involve the following cascade of signal transduction reaction: Bradykinin combines with its B2 receptor, causes extracellular Ca2+ influx, in-tracellular Nitric Oxide and cyclic GMP increase, enhances formation of transen-dothelial pinocytotic vesicles and finally opens the BTB. This pathway is in no sense complete;many links remain to be found. More recently, ATP - sensitive potassium (KATP) channel was reported involving in brain tumor microvessel permeability regulation and increasing the BTB permeability. We suppose thatKATP channel serve as an important target in the biochemical regulation of BK -induced BTB permeability increase. Experiments are dedicated to investigate this possibility.The current study includes establishment of rat brain C6 tumor model, testing whether or not intracarotid BK infusion induces overexpression of Kir6. 2 subunits on tumor cells and tumor capillaries in tumor model and testing whether or not BK causes the Kir6. 2 subunit distribution change in tumor sites before and after infusion. It also investigates whether or not BK mediate the KATP channels opening on cultured C6 tumor cells. This study tries to analyse the relationship between upregulation of KATP channel and BK perfusion, and aims at elucidating the role of KATP channels in BK - induced BTB permeability modulation and further delineating the operative mechanism. This study may have significant implications for improving the targeted delivery of anti - neoplastic agents to brain tumors and may provide new therapeutic target and new method for treatment of central nervous system diseases.Materials1. Experimental animals: The adult female Wistar rats (180 -200 g) were used, which purchased from the Center for Experimental Animals of China Medical University. The total number was 72. All animal experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.2. Experimental cell line: The C6 glioma cell line was donated by Department of Neurosurgy in Yamanashi University, Janpan.3. Experimental reagents-, rabbit polyclonal antibody anti - kir6. 2 and mouse monoclonal anti - vWF primary antibody ( Santa Cruz Biotechnology, Inc. );Bradykinin (BK) , ECL (enhanced chemiluminescence) , anti -rabbit IgG conjugated with FITC, anti - mouse IgG conjugated with CY3, Minoxidil Sulfate (MS) and Glibenclamide (Sigma - Aldrich, Inc. );goat anti -rabbit antibody conjugated with horseradish peroxidase and SABC immunohistochemis-try kit (Zhongshan Company, Beijing);K2ATP (Sigma - Aldrich, Inc. ).4. Experimental instruments: C02 incubator, superclean bench, ster-eotaxic apparatus for rat brain, low temperature refrigeration centrifuge, ultraviolet spectro photometer, automatic running gel imaging system, GP electropho-resis apparatus, upright fluorescence microscope, - 80 Tl deep freeze refrigerator , homeothermia freezing microtome, Motic Images Advanced 3.2 image analysis system. PP -83 puller, Axopatch - ID patch - clamp amplifier, VC - 10, AD/DA conversion plate and PC computer fitted with pCLAMP 5.5.1 software.Methods1. Establishment of rat brain C6 tumor model.2. Make sure the location of transplanted glioma in rats brain and the pathological grade with hematoxylin and eosin (H&E) method.3. After low dose of BK was pumped into rat' s brain with glioma via the carotid artery, the expressions of KATP channel protein in tumor were determined with Western blot at perfusion 5min, lOmin, 15min and 30min. MS, KATP channel ' s agonist, was administrated to gloma - rats and BK coinfusing with KATP channels antagonist Glibenclamide was also administrated. The induced expressions of KATP channel protein by MS and BK plus Glibenclamide compared with what induce by BK infusion.4. According to the results of H&E, the KATP channels expression level and distribution changes induced by low dose of BK was investigated with Immuno-histochemistry.5. Fluorescence microscopes analysis was used to detect colocalization and expression of KATP channels in brain tumor capillary endothelium and tumor tissues , both before and after low dose of BK infusion.6. Observation of IKATP induced by BK and KATPchannel's antagonist in C6 tumor cells with Patch - clamp technique in Whole - cell configuration.7. Image analysisThe data obtained from Western blot were scanned with Chemi Imager 5500 V2.03 software;Integrated Density Value (IDV) of the KATP channels was calculated with Fluor Chen 2. 0 software. The data of Immunohistochemistry wereobtained and analysed semiquantitatively with Motic Images Advanced 3. 2 image analysis system. The data of Immunolocalization of KATP channels were analysed with SPOT Advanced 4. 0.2 software.8. Statistics analysisThe experimental data were calculated with SPSS (statistical package for the social science). Results are expressed as the mean SD. For immunoblot a-nalysis, one - way analyses of variance (ANOVA) were used to compare group differences in measurement of kir6.2 subunits. Dunnetts post hoc tests were applied to compare specific group difference if the ANOVA revealed a significant difference. In other measurements, the data were assessed using paired Student' s t test. P < 0.05 was considered statistically significant.Results1. Results of H&E analysisIn 72 rat' s brains with glioma cells implantation, the tumors were found in the head of caudate nucleus of right brain, the pathological grades were M. The contralateral brain with tumor cells implatation was served as control group.2. Results of Western blot analysisCompared with control group, the expression of Kir6.2 subunits in tumor -bearing rat brain increased remarkably after 5 minutes of BK infusion. At administration 10 min, a significant increase of IDV ratio of Kir6.2 subunits was noted, which was 73. 58% comparing with control group. After that the expression decreased gradually, 15min after stop of BK infusion ( BK 30min group) , the expression was nearly identical to that in the control group. After lOmin of MS infusion, the expression of Kir6. 2 subunits was increased dramatically, much more than the BK lOmin group. And Glibenclamide significantly inhibited Kir6. 2 subunits expression. The integrated density value (IDV) ratio of Kir6. 2 sub-units to p - actin in control group was 0. 511 ± 0.037 (n =â– 8). The IDV ratios of Kir6. 2 subunits to p - actin in experimental groups were 0.766 ± 0. 058 (BK5min, n = 8, P < 0.05), 0.887 ± 0.065 (BK lOmin, n = 8, P < 0.01), 0.727 ± 0.055 (BK15min, n = 8, P < 0.05) and0.636 ± 0.045(BK30min, n = 8, P < 0.05) respectively. The IDV ratios of Kir6.2 sub-units to (3 - actin in MS group and in BK plus Glibenclamide group were 1.177 ± 0.092 (perfusion lOmin, n = 8, P < 0.01) and 0. 590 ± 0.041(perfu-sion lOmin, n = 8, P < 0.05) , individually.3. Results of Immunohistochemistry analysisIn tumor tissues with sterile saline, Kir6. 2 subunits expressed slightly, mainly on the tumor capillaries and tumor cells. After 10 min bradykinin infusion, Kir6. 2 - like immunoreactivity was found on the tumor capillaries and tumor cells, in areas surrounding tumor capillaries, the majority of the Kir6. 2 subunits appeared on cell membranes. BK lOmin group had a significant increase in Kir6.2 - like immunoreactivity. The mean optical density value was 0. 219 ±0.01, higher than the value of 0.184 ±0.009 (n = 8) in control group. P < 0. 01 was considered statistically significant.4. Results of Fluorescence microscopes analysisThe Immunolocalization of ATP - sensitive potassium ( KATP ) channels (green) and von Willebrand factor (red) were also observed with fluorescence microscopes before and after BK administration. The yellow areas are KATP channels colocalizing with vWF on capillary endothelial cells. After lOmin of BK infusion , different expressions of Kir6. 2 subunits were found among different - diameter microvessels. Abundant expressions of KATP channels were observed on tumor capillries of lOfxm in diameter, while the expressions on 40jxm - diameter microvessels were similar to the control.5. Results of Patch - clamp technique in Whole - cell configuration When the holding potential was at - 40 mV, the command potential was-60 to +60 mV, and the intrapipette solution contained 0. 3 mM NajATP. An outwardly potassium current was recorded, which could be inhibited by KATP channels antagonist, glibenclamide (10 jxmol/1). It was an indication that the outwardly potassium current was I KATP. During the lOmin observation, the current was kept at a stable level. The current densities (pA/pF) of I ^p increase with the increasing of command potentials, when command potential ranging from -60 to +60 mV, the concentrations of bradykinin being 1 x 10 8, 1 x 10 , 1 xlO" mol/1, respectively, and Bradykinin perfusion time being lessthan 1 min. When command potential was at + 60 mV, the amplitude values of IKATP were increased from 51. 50 ± 4. 63 pA/pF ( of control) , to 53. 97 ±3.16 pA/pF (n=8, P>0.05), 64.79 ± 7. 73 pA/pF (P >0.05) and 67. 53 ± 6. 19 pA/pF (P >0. 05) , respectively. Hence, if the bradykinin perfusion times was less than 1 min, the amplitude values of I KATP were statistically insignificant , even if the concentration of BK was at the most appropriate value of 1 xlO"6mol/l.When bradykinin perfusion time was longer than 5 min (concentrations was1 jxmol/1) , the current densities (pA/pF) of I^p was higher. Therefore, we recorded current densities ( pA/pF) of IKATP at perfusion 5 , 10, 15 and 30min to investigate the electrophysiological changes of KATPchannels in response to BK. When BK perfusion time was lOmin, the slope of current - voltage curve was the highest. When the command potential was at + 60 mV, amplitude of IKATP were 57.49 ±3.13 pA/pF of control, 71.29 ±4.96 pA/pF (n=8, P<0.05), at perfusion5 min;85.49 ±7.35 pA/pF (P<0.01) , at perfusion 10 min;68.99±5.69 pA/pF (P<0.05) , at perfusion 15 min;44.90±5.91 pA/pF (P<0. 01), at perfusion 30 min. The amplitude of IKATP with Glibenclamide (10 /xmol/ L) was 36. 80 ±6.77 pA/pF.DiscussionThe current experiment shows that BK could modulate the expression of KATP channels in a time - associated manner, with peak expressions at 10 minutes of BK perfusion. A significant increase of 73. 58% , of IDV ratio of Kir6.2 subunits is observed. The increased KATP channels are mainly located on tumor capillaries of 10 jim in diameter. The overexpression of KATP channel achived by BK was significantly attenuates when confuses with antagonist, Glibenclamide. BK significantly increases IKATP on cultured C6 glioma cells in a time - dependent manner;the slope of current - voltage curve at BK lOmin perfusion was the highest. All the above - mentioned results strongly suggeste that BK - mediated BTB permeability increasing is associated with KATP channels, which are important targets in the biochemical regulation of BTB permeability increase with BK.The blood - brain barrier ( BBB) is a very effective obstacle, which stops most of neurotherapeutics to be delivered to brain. When the patients were suffering from the brain glioma, however, BBB still inhibits the delivery of most anticancer drugs, especially at the margin of proliferated tumors. In order to delivery the anti - tumor drugs to brain efficiently, many investigations were done to explore the method of selectively opening BBB. K. L. Black first reported that low dose of Bradykinin could transiently increase the permeability of the BTB without normal brain involvement. But in clinical trial, the BK analogRMP -7 did not produce an ideal therapeutic effect hence limited the use of BK as a reliable BBB opener. Lot's of investigations were done to explore the underlaying mechanism. The current study shows that Bradykinin enhances KATP channes expression in a rat brain ( C6) glioma model and significantly increases Iratp on cultured C6 glioma cells in a time - dependent manner. We suggested that KATP channes could regulate BK - induced BTB permeability. Based on the results, we suggest the mechanism of BK - induced BTB permeability increase could be complemented as following;BK diffuses through the intact capillary endothelium of glioma or directly cross the broken tumor capillary endothelium;then combines with B2 receptors on tumor cells to produce endogenous NO. The endogenous NO reversely diffuses into capillary endothelium to active sGC causing intr-cellular cGMP upregulation, then active KATP channels to increase transendo -thelial pinocytotic vesicles, which can transport drugs across BTB. At the same time, the NO - cGMP system in glioma cells is activated to modulate KATP channels for increasing formation of pinocytotic vesicles to delivery therapeutics.BK - induced BTB permeability increase is transient and fluctuant, lasting about 20 minutes;the maximum degree of opening appeared at perfusion 10 min. In our study, the up - regulation of KATP channels modulated by BK is less than 30 min and the maximum value of expression is also observed at BK perfusion 10 min. The coincidence implies that KATPchannels are the downstream biological targets responsed to BK following combined with B2 receptors to open BTB. Wang and Xue found that most B2 receptors on glioma cell membranes were internalized into the cytoplasm at 10 minutes of BK infusion,which strongly supported our estimation.However, some studies have shown that Bradykinin' s analog, Cereport, could increase the delivery of pharmacological agents across the normal (i. e. nontumor) BBB without B2 receptors involving. It is a challenge to explain this delivery. According to our observation, BK directly activates KATPchannels in rat glioma cells, and the same KATPchannels present on capillary endothelium, Bradykinin may directly open KATP channels on normal capillary endothelium to increase the permeability. We also observed that the up - regulation of KATP channel prtein expression appeared in normal brain tissues after 15 minutes of BK perfusion and the peak also rearched after 10 - minute perfusion;and Schnitzler demonstrated a signal pathway that maybe also closely linked to our suggestion.All in one sentence, the current study demonstrated that KATP channels are important targets in the biochemical regulation of BTB permeability increase induced by BK. In clinical trail, we could use KATP channels agonist as a substitute for Bradykinin or its analog to selectively open BTB in human glioma, and therefore improve the curative effects of glioma and life quality of patients with glioma.Conclusion1. The current study for the first time demonstrates that an enhanced expression of KATP channel on tumor sites of rat brain glioma (C6) model could be achieved after Bradykinin infusion;this effect is especially obvious at 10 minutes of administration.2. The overexpressions of KATP channel most exist on the tumor capillaries of 10(Am in diameter after BK infusion, while is barely detectable on tumor capillaries with 40 (jim diameter.3. BK could open KATP channel on cultured C6 gliomas cells in a time -dependent manner;this effect is especially obvious at 10 minutes of administration.
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