Mechanism Of Combination Of Low-frequency Ultrasound And Low-dose Bradykinin In Opening Blood-tumor Barrier

ObjectiveBlood-tumor barrier(BTB),similar to blood-brain barrier(BBB),is located between glioma cells and macrovessels formed by highly specialized endothelial cells. It restricts the paracellular diffusion of hydrophilic molecules to tumor tissue by an elaborate network of complex endothelial tight junction(TJ),which contains several ubiquitous molecular components including claudins,occludin,ZO-1,ZO-2,and ZO-3. The permeability of BTB is slightly higher than that of BBB,but it is still a significant barrier for therapeutic drugs to penetrate.Various approaches have been applied to increase the permeability of BTB in chemotherapy.However,these approaches are restricted from wide clinical use because of the high dosage,invasiveness,low reliability,or short opening time of the BTB.Low-frequency ultrasound(LFU) is one of the routine diagnostic methods.LFU could penetrate through the tissue easily,is lower to be absorbed of power,and causes less damage to the tissue,so it has been used in experimental therapy in recent years. LFU has been applied to osteoarthropathy,infection,inflammation,fracture, thrombolysis,parenchyma repairment,and anti-tumor therapy.Recently a few studies have shown that LFU can open brain BBB,with frequency from 260 kHz to 1.63 MHz, and the highest frequency is 3.5 MHz.LFU increases the penetration of durgs after opening BBB,but the standard parameters of ultrasound criteria have not been obtained. In our previous study,BBB permeability increased after LFU sonication(frequency 1 MHz,power 12 mW) without detectable neuron damage.Therefore,LFU may be used to open BTB in chemotherapy.The mechanism of the effect of LFU on BTB permeability is still unclear.Low-dose bradykinin(BK) transiently increases the permeability of BTB, selectively opens BTB,possibly through its interaction with BK type 2(B2) receptor on glioma cells,and increases the transportation of carboplatin into brain tumors, without the obvious vasodilatory effect caused by high-dose BK.Low-dose BK infusion may prolong the survival time in rats with gliomas.However,the clinical use of low-dose BK is also limited because of a fast enzymatic degradation.The purpose of this study was to determine whether LFU and low-dose BK increase the permeability of BTB in a synergistic manner and whether TJ is involved in BTB permeability in C6 glioma rats.We examined the effects of LFU on BTB permeability,and determined the expressions of TJ-related proteins,claudin-5,occludin, and ZO-1.Methods1.C6 brain glioma rats model were established.2.Before and after LFU sonication and BK infusion individually or in combination,the BTB permeability was determined by Evans blue(EB) assay in vivo in rats with C6 brain glioma.3.Before and after LFU sonication and BK infusion individually or in combination,the changes in TJs and ultrastructure were observed under transmission electron microscope in BTB model in vivo in rats with C6 brain glioma.4.Before and after LFU sonication and BK infusion individually or in combination,RT-PCR were used to detect the protein expression levels of TJ associated proteins,elaudin-5,occludin,and ZO-1,in BTB model in vivo in rats with C6 brain glioma.5.Before and after LFU sonication and BK infusion individually or in combination,immunohistochemistry was performed to detect the distribution and expression of TJ associated proteins,claudin-5,occludin,and ZO-1,in BTB model in vivo in rats with C6 brain glioma. 6.Before and after LFU sonication and BK infusion individually or in combination,immunofluorescence was used to detect the distribution and expression of TJ associated proteins,claudin-5,occludin,and ZO-1,in BTB model in vivo in rats with C6 brain glioma.7.Before and after LFU sonication and BK infusion individually or in combination,Western blot and RT-PCR were performed to detect the protein expression levels of TJ associated proteins,ZO-1,occludin,and claudin-5,in BTB model in vivo in rats with C6 brain glioma.Results1.In C6 glioma rats,the permeability of BTB increased after LFU sonication, reached the peak at 1.5 h,and recovered 12 h later.2.In C6 glioma rats,TJ was opened by LFU,and the apoptosis of C6 glioma cells was induced,without neuronal injury.3.In C6 glioma rats,mRNA expression of TJ related proteins,claudin-5, occludin,and ZO-1,significantly decreased,reached the minimum at 1.5 h,and recovered 12 h later.4.In C6 glioma rats,protein expression of TJ related proteins,claudin-5, occludin,and ZO-1,significantly decreased,reached the minimum at 1.5 h,and recovered 12 h later.5.The permeability of BTB significantly increased after the individual application of LFU and BK,and further increased after LFU applied in combination with BK.6.TJ opening was observed under transmission electron microscope after LFU and BK applied individually and in combination.7.The mRNA expression of TJ related proteins,claudin-5,occludin,and ZO-1, significantly decreased after the application of LFU and BK individually,and further decreased after LFU applied in combination with BK.8.The protein expression of TJ related proteins,claudin-5,occludin,and ZO-1, significantly decreased after the application of LFU and BK individually,and further decreased after LFU applied in combination with BK. DiscussionIn the study,we proved that LFU can increase the permeability of BTB and open the BTB reversibly.No neuronal injury was observed,but the apoptosis of C6 glioma cells was induced during the sonication.The permeability of BTB significantly increased and TJ opening of BTB was observed by the combination of LFU and BK. Additionally,the mRNA and protein levels of TJ-related proteins,claudin-5,occludin, and ZO-1,reduced significantly.Furthermore,the permeability of BTB significantly increased and the expressions of TJ-related proteins,claudin-5,occludin,and ZO-1, significantly decreased in 5/6LFU+2/3BK and LFU+BK groups compared with LFU and BK groups.The results in this study indicate that the combination of LFU and low-dose BK increase the permeability of BTB in a synergistic manner in the rat model of C6 glioma,and TJ opening may contribute to the LFU/BK-induced increase in BTB permeability.The low frequency(1 MHz) and low pressure(0.48 MPa) ultrasound we used here is the same as that used in our previous study.The increased EB content after LFU sonication,indicating an increase in BTB permeability,confirmed our previous results. Different powers and pressures of LFU were also reported to open BBB locally and increase the permeability of agents and antibodies through BBB.Targeted delivery of doxorubicin and gadolinium into the brain was successfully enhanced by LFU with a frequency of 1.5-1.7 MHz and pressure amplitudes of 0.67-0.8 MPa,without a significant increase in brain temperature.However,brain tissue necrosis,neuron apoptosis,and red blood cell extravasation in the sonicated area have been reported after LFU sonication at relatively high pressures and frequencies(frequency of 690 kHz and pressure amplitude of 2.3 to 3.1 MPa or a frequency of 2.04 MHz and pressure amplitude of 0.3 to 2.3 MPa) by Hynynen et al.and McDannold et al.The low frequency and low pressure used in this study,which achieved a similar effect on BTB to other studies without detectable neuron damage,might reduce the adverse effect of LFU sonication and be applied clinically as a feasible approach in tumor treatment.The exact mechanism for LFU-induced increase of BTB permeability is unclear. LFU may alter BBB permeability by modifying cerebral blood flow through vasoconstriction,or by inertial cavitation.Besides,TJ is a stable structure to keep dynamic equilibrium of paracellular permeability and may be disorganized and reorganized quickly by extracellular stimulations.In this study,openings of TJ were found between brain microvessel endothelial cells and tumor tissues in all experimental groups,indicating that TJ opening is indeed involved in the increase of BTB permeability and the paraendothelial route may be one of the mechanisms for the increase of BTB permeability by LFU,as suggested in Hynynen et al.The reduction in TJ-related proteins,claudin-5,occludin,and ZO-1,in brain microvessels after LFU sonication confirmed the important role of TJ opening in BTB permeability.The increase in BTB permeability and down-regulation of TJ-related proteins after low-dose BK infusion are consistent with our previous study,indicating a paraendothelial route by BK.Other mechanisms may also be involved,such as the opening of ATP-activated(K_ATP) or Ca²⁺-activated(K_Ca) potassium channels.Despite the obvious effect of low-dose BK on BTB permeability,the duration of BK-induced increase in BTB permeability is usually transient,with the peak at 15 to 30 min and full recovery within 60 min.The clinical application of BK as a BTB opening substance is limited partly because of its transient effect.Since individual usage of either LFU or BK has noticeable limitations,we investigated the combinatory effect of LFU and low-dose BK in this study.To our knowledge,the combination of LFU and low-dose BK has not been reported.Both 5/6LFU+2/3BK and LFU+BK treatment induced a more potent increase in BTB permeability and reduction in ZO-1,occludin,and claudin-5 expressions than LFU and BK individually.Therefore,we conclude that LFU and BK act in a synergistic manner to increase the BTB permeability when used in combination.Furthermore,as there was no significant difference between 5/6LFU+2/3BK and LFU+BK groups,the dose of 5/6LFU+2/3BK may be used as an optimal dose for this combined treatment.It is likely that the low-dose combination may lower the currently reported side effects of LFU and BK individually.In addition,the considerably prolonged duration on the increase of BTB permeability by 5/6LFU+2/3BK(see results section) also favours a wider use of the combined application of LFU and BK.In summary,this study demonstrates that the combination of LFU and BK at low dose exerts a synergistic effect on increasing BTB permeability,possibly through a paraendothelial route.This finding may provide some new guidance to find a clinically applicable method to locally open BTB and deliver anti-tumor agents to tumors.Conclusions1.The optimum parameters for LFU to open BTB are frequency of 1 MHz, power of 12 mW,pressure of 0.48 MPa and exposure time of 20 s.2.LFU can increase the permeability of BTB and open BTB reversibly,inducing the apoptosis of C6 glioma cells and doing no harm to neurons.3.LFU can reduce the expression of TJ related proteins of claudin-5,occludin and ZO-1,at mRNA and protein levels,and TJ opening may be related to the effect induced by LFU.4.LFU and BK increase the permeability of BTB in a synergistic manner in the rat model of C6 glioma,and TJ opening may contribute to the LFU/BK-induced increase in BTB permeability.5.There was a further decrease in TJ related proteins of claudin-5,occludin,and ZO-1 reduced by LFU and BK in a synergistic manner.