| Background:Cervical cancer is one of the most common malignant tumors in gynecological tumors,and cisplatin-based concurrent chemoradiation is one of standard treatment options for it.Cisplatin is a widely used dose-dependent chemotherapeutic drug,and its severe toxic and side effects restrict its clinical dosage.This study intends to enhance the concentration of cisplatin by sonophoresis to achieve the goal of improving local tumor control.Methods:Firstly,a xenograft tumor model was successfully established under the skin of nude mice with cervical cancer Si Ha cell line.Secondly,sonophoresis was used to transdermal different concentrations cisplatin into the tumor tissue and the related safety were evaluated.Finally,the relevant mechanisms of sonophoresis were researched,and the safety and feasibility of combined with conventional chemotherapy in the treatment of cervical cancer were evaluated.The specific research methods were as follows:1.The nude mice were randomly divided into a control group and an experimental group,with 8 mice in each group.The control group was fed normally without any intervention,and the experimental group was injected subcutaneously with 2×10~6 Si Ha cells per mouse.The tumor and the general condition of mice were observed.After the tumor grow up,sacrificed the mice and check the damage and metastasis of normal organs.2.Fifty-six female nude-mice model of cervical cancer were randomly divided into 7 groups(n=8 in each group):control group without any intervention,sonophoresis 0.2mg/ml group,sonophoresis 0.4mg/ml group,sonophoresis 0.8mg/ml group,plaster therapy 0.2mg/ml group,plaster therapy0.4mg/ml group and plaster therapy 0.8mg/ml group.Sonophoresis groups were treated with the corresponding concentrations of cisplatin respectively with concurrent sonophoresis applied on the skin of local tumor,1ml at a time,once a day for a total of 5 days(Therapeutic pulsed ultrasound was 1.0MHz,2.0W/cm~2and 60 minutes duration).Plaster therapy groups use the same way of administration except the therapeutic ultrasound.Weight of mice and tumor diameters were measured every day during the intervention.The concentration of cisplatin in tumors was detected by HPLC.According to the results of HPLC,the tumors,skins,livers and kidneys gross structures and ultrastructure of sonophoresis groups were observed in order to evaluate the effectiveness and safety of experimental conditions.In addition,apoptosis and proliferation-related factors(MPO,Caspase-3,PCNA)were detected by immunohistochemistry,immunofluorescence and TUNEL assay.3.Thirty-two female nude-mice model of cervical cancer were randomly divided into 4 groups(n=8 in each group):control group without any intervention,the intraperitoneal group:intraperitoneal injection of cisplatin 5mg/kg,once a day for 5 days,the sonophoresis group treated with the cisplatin concentration 0.4mg/ml under sonophoresis on the skin of local tumor 1ml once a day for 5 days,and therapeutic pulsed ultrasound was 20 minutes duration,frequency of 1.0MHz,intensity of 2.0W/cm~2.The combination group treated with intraperitoneal injection and sonophoresis of cisplatin.Organ damage was evaluated by HE stains and TEM.Apoptosis was assessed by TUNEL assay,and transmission electron microscopy assay.Relative quantitative real-time PCR and Western blot were used to determine the expressions of genes and proteins.Results:1.After 14 days of injection of Si Ha cells,a model of cervical cancer xenograft in nude mice was successfully established,with a tumor volume of 100-200 mm~3.The experimental group had no changes in living habits versus to the control group.And there was no difference in body weight between the two groups.Cervical cancer xenografts were confined to under the skin,no systemic lymph node metastasis was observed,and lung,liver,kidney,and spleen tissues were normal,without metastases.2.The weight of nude mice in each group showed an increasing trend,except for a decrease of weight in the sonophoresis 0.8 mg/ml group.No obvious tumor inhibition effect was observed.Cisplatin was detected in the sonophoresis 0.4 mg/ml group and sonophoresis 0.8 mg/ml group,with relative concentrations of 0.081±0.033 mg/ml,0.111±0.021 mg/ml,respectively.No cisplatin was detected in other groups,so no further detection was conducted in plaster therapy groups.Both skin and kidney inflammation were observed in sonophoresis 0.8 mg/ml group.In sonophoresis groups,the expression of MPO,caspase-3 and TUNEL showed a concentration dependent,and the expression increased with the increase of the guided drug concentration.The expression of PCNA was opposite.3.Cisplatin transdermal by sonophoresis inhibited the growth of cervical cancer xenografts,without obvious toxic and side effects on local skin,liver,kidney,lung and spleen tissues.It increased the tumor suppression rate combined with intraperitoneal injection of cisplatin.Meanwhile,there was no significant difference of organ indexes between the combination group and the intraperitoneal injection group.Cisplatin transdermal by sonophoresis activated the JNK and p38 signaling pathways in the MAPK pathway,inhibited the activation of ERK pathway,improved the expression of p53 in tumor tissues,reduced the ratio of Bcl-2/Bax,and increased the activation of Caspase-9 and Caspase-3,thus induced the tumor apoptosis and inhibiting the proliferation of transplanted tumors.Conclusions:1.Subcutaneous injection of 2×10~6 Si Ha cells per mouse can successfully establish a model of cervical cancer xenograft in nude mice,without obvious adverse effects on nude mice.2.Sonophoresis enhanced transdermal delivery of cisplatin in a xenograft tumor model of cervical cancer.3.Considering the occurrence of skin inflammation and renal injury caused by cisplatin,the recommended concentration to sonophoresis is 0.4mg/ml.4.Cisplatin transdermal by sonophoresis can inhibits growth of cervical cancer in vivo without any side effect.Combined with intraperitoneal injection of cisplatin,it increased the tumor inhibition rate and did not increase any liver or kidney damage.5.Apoptosis pathways were confirmed by modulation of p53 and Bcl-2family members.6.The MAPK pathway might be one of the related pathways.Sonophoresis drug transdermal delivery,as an adjuvant administration method combined with conventional chemotherapy enhanced the tumor inhibition rate without increasing the side effect. |
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