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Objective To assess the effect of fenretinide (4-HPR) on proliferation,apoptosis and migration of B16F10 and A375 melanoma cells,and to evaluate the effect of liposomes and RGD peptidemodified liposomes on its uptake and therapeutic effects.Methods A film-hydration method was used to prepare 4-HPR liposomes (4-HPRL),which were modified with RGD peptide to prepare RGD-4-HPRL,and the concentration,particle size,electric potential,drug loading capacity and encapsulation efficiency were measured for 4-HPRL and RGD-4-HPRL.In vitro cultured B16F10 and A375 cells were divided into several groups:4-HPR group,4-HPRL group and RGD-4-HPRL group treated with Dulbecco's minimum essential medium (DMEM) containing 4-HPR bulk drug,4-HPRL and RGD-4-HPRL respectively at the same concentration of 4-HPR,and control group treated with culture solution at the same volume.After different durations of treatment,cell counting kit-8 (CCK8) assay was performed to evaluate cellular proliferative activity,annexin V/propidium iodide staining to detect apoptosis,and scratch wound healing assay to evaluate the effect of drug treatment on cell migration ability.Then,4-HPR was replaced by coumarin 6 (C6) to prepare C6 liposomes (C6L) and RGD-C6L,and flow cytometry was conducted to evaluate C6 uptake by B16F10 cells.Statistical analysis was carried out with SPSS22.0 software by one-way analysis of variance (ANOVA) for the comparison among several groups and t test for the comparison between two groups.Results The concentration of 4-HPR in the prepared 4-HPRL solution was over 1 300 mg/L.The encapsulation efficiency and drug loading capacity of 4-HPRL were (95.51 ± 1.22)％ and (7.27 ± 0.11)％ respectively,and those of RGD-4-HPRL were (95.82 ± 0.81)％ and (7.14 ± 0.13)％ respectively.The particle size distribution of 4-HPRL and RGD-4-HPRL was uniform,and their average particle size was below 100 nm.CCK8 assay showed that 4-HPR could markedly inhibit the proliferative activities of B16F10 and A375 cells.The cell proliferation inhibition rate of 4-HPRL was higher than that of 4-HPR at the same concentration of 4-HPR (P ＜ 0.01),and the inhibition rate of RGD-4-HPRL was higher than that of 4-HPRL (P ＜ 0.01 or P ＜ 0.05) and 4-HPR (P ＜ 0.01).As annexin V/propidium iodide apoptosis assay showed,when the concentration of 4-HPR was 10 mg/L,the total apoptosis rates of B16F10 cells in the control group,4-HPR group,4-HPRL group and RGD-HPRL group were (4.44 ± 0.35)％,(28.33 ± 0.66)％,(46.43 ± 0.77)％ and (51.33 ± 0.37)％ respectively.When the concentration of 4-HPR was 20 mg/L,the total apoptosis rates of A375 cells in the above 4 groups were (4.97 ± 0.62)％,(16.68 ± 3.81)％,(32.62 ± 1.24)％ and (44.85 ± 4.92)％ respectively.The apoptosis rates of B16F10 and A375 cells were significantly higher in the 4-HPRL group than in the 4-HPR group (both P ＜ 0.01),and higher in the RGD-4-HPRL group than in the 4-HPRL group (both P ＜ 0.01) and 4-HPR group (both P ＜0.01).Scratch wound healing assay showed that 4-HPR could inhibit scratch healing and migration of B16F10 and A375 cells,and the inhibitory effects of 4-HPRL and RGD-4-HPRL were distinctly superior to those of 4-HPR bulk drug.C6 uptake assay revealed that the fluorescence intensity of C6 in B16F10 cells in the control group,C6 group,C6L group and RGD-C6L group were 2.15 ± 0.28,8.56 ± 0.36,20.48 ± 0.13 and 22.55 ± 0.07 respectively,and there were significant differences between the 4 groups (F =67 194.186,P ＜ 0.01).Additionally,the fluorescence intensity of C6 was significantly higher in the C6L group and RGD-C6L group than in the C6 group (both P ＜ 0.01),and higher in the RGD-C6L group than in the C6L Group (P ＜ 0.01).Conclusions 4-HPR can inhibit the proliferation and migration of A375 and B16F10 cells,and induce their apoptosis.Liposomes and RGD-targeted liposomes can markedly enhance the effect of 4-HPR on melanoma cells.
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Objective@#To explore the effects of N-(4-hydroxyphenyl) retinamide (4HPR), 4HPR liposome (4HPR-L), and 4HPR lipid microbubble (4HPR-LM) combined with ultrasound on proliferation, apoptosis, and cell cycle of human keloid fibroblasts (Fbs).@*Methods@#(1) 4HPR-L and 4HPR-LM were prepared by hydration ultrasonic method. The appearance morphology, particle size distribution, Zeta potential, loading drug concentration, encapsulation efficiency, and drug loading rate of 4HPR-L were investigated by high performance liquid chromatography, dynamic light scattering, and transmission electron microscope. (2) Human keloid Fbs were cultured and divided into 13 groups by random number table (the same grouping method below), with 6 wells in each group. Cells in control group were given no treatment, while cells in 12 ultrasound groups including 0.5 W 30 s group, 0.5 W 60 s group, 0.5 W 120 s group, 0.7 W 30 s group, 0.7 W 60 s group, 0.7 W 120 s group, 1.0 W 30 s group, 1.0 W 60 s group, 1.0 W 120 s group, 1.5 W 30 s group, 1.5 W 60 s group, and 1.5 W 120 s group were treated by ultrasound with corresponding parameters. The cells viability was measured by a microplate reader after 24 hours of routine culture. Another batch of human keloid Fbs were divided into 5 groups, with 6 wells in each group. Cells in control group were given no treatment, while cells in 1, 10, 20, and 50 μg/mL blank lipid microbubble groups were treated with blank lipid microbubbles in corresponding mass concentration. The cells viability was measured as before after 24 hours of routine culture. Another batch of human keloid Fbs were divided into 6 groups, with 12 wells in each group. Cells in control group were given no treatment, while cells in 1, 10, 20, 50, and 100 μg/mL 4HPR-L groups were added with 4HPR-L carrying corresponding mass concentration of 4HPR. The cells viability in 6 wells of each group was detected after 24 and 48 hours of routine culture, respectively. Another batch of human keloid Fbs were divided into 4 groups, with 6 wells in each group. Cells in control group were given no treatment, while cells in 4HPR, 4HPR-L, and 4HPR-LM+ ultrasound groups were treated with 4HPR, 4HPR-L, and 4HPR-LM (all the mass concentration of 4HPR was 20 μg/mL), respectively, and cells in 4HPR-LM+ ultrasound group were given 0.5 W 60 s ultrasound treatment immediately after drug administration. The cells viability was measured as before after 24 hours of routine culture. (3) Another batch of human keloid Fbs were divided into control group, 4HPR group, 4HPR-L group and 4HPR-LM+ ultrasound group, with 3 wells in each group, and the cells in each group were treated as before. Apoptosis of the cells was detected by flow cytometer after 24 hours of routine culture. (4) Another batch of human keloid Fbs were grouped and treated as in (3), and then the cell cycle distribution was detected by flow cytometer after 24 hours of routine culture. Data were processed with one-way analysis of variance and t test.@*Results@#(1) 4HPR-L particles had a spherical or spheroidal structure and were uniform in size, with particle size of (100.1±1.3) nm and Zeta potential of (-34.3±2.3) mV. The mass concentration of 4HPR in 4HPR-L solution was about 1 400 μg/mL, with the encapsulation efficiency of (95.8±1.2)% and drug loading rate of (8.3±0.4)%. (2) The viability of cells in the 12 ultrasound groups was higher than 93.0%, and the viability of cells in 1, 10, 20, and 50 μg/mL blank lipid microbubble groups was higher than 95.0%. The viability of cells in 1 μg/mL 4HPR-L group at administration hour 24 was similar to that at 48 (t=0.393, P>0.05). The viability of cells in 10, 20, 50, and 100 μg/mL 4HPR-L groups at administration hour 24 was significantly higher than that at administration hour 48 (t=44.593, 22.961, 32.224, 35.337, P<0.01). The viability of cells in 4HPR group, 4HPR-L group, and 4HPR-LM+ ultrasound group was (47.3±0.7)%, (42.3±1.7)%, and (38.6±0.8)%, respectively. The viability of cells in 4HPR group was significantly higher than that in 4HPR-L group and 4HPR-LM+ ultrasound group (t=4.551, 15.895, P<0.05 or P<0.01). The viability of cells in 4HPR-L group was significantly higher than that in 4HPR-LM+ ultrasound group (t=-3.360, P<0.05). (3) The percentages of total apoptotic cells in 4HPR group, 4HPR-L group, and 4HPR-LM+ ultrasound group were (32.8±2.4)%, (42.5±2.4)%, and (58.5±6.3)%, respectively, which were significantly higher than the percentage of control group [(14.9±1.6)%, t=8.748, 13.637, 9.500, P<0.01]. The percentages of total apoptotic cells in 4HPR-L group and 4HPR-LM+ ultrasound group were significantly higher than the percentage in 4HPR group (t=4.049, 5.393, P<0.05 or P<0.01), and the percentage of total apoptotic cells in 4HPR-LM+ ultrasound group was significantly higher than that in 4HPR-L group (t=3.371, P<0.01). (4) The percentage of G2/M phase cells in 4HPR group was higher than that in control group, but there was no statistically significant difference (t=2.107, P>0.05). The percentage of G2/M phase cells in 4HPR-L group was significantly higher than that in 4HPR group or control group (t=18.169, 30.026, P<0.01). The percentage of G2/M phase cells in 4HPR-LM+ ultrasound group was significantly higher than that in 4HPR-L group, 4HPR group, and control group (t=4.932, 25.854, 66.231, P<0.01).@*Conclusions@#4HPR can inhibit proliferation, induce apoptosis, and arrest G2/M phase of human keloid Fbs, and the effects of 4HPR-LM combined with ultrasound are better than those of 4HPR-L and free 4HPR.
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AIM:To investigate the clinicopathological significance of the protein expression of phosphorylated ezrin at threonine 567 ( pEZRThr567 ) in lung squamous cell carcinoma, adjacent tissues and normal tissues.METHODS:pEZRThr567 protein was detected in lung squamous carcinoma, adjacent and normal tissues by the method of immunohisto-chemistry.The correlation of pEZRThr567 expression with clinicopathological parameters of lung squamous carcinomas was al-so analyzed.The localization of pEZRThr567 was detected by immunofluorescence staining in lung squamous cell line EBC-1. RESULTS:The protein expression of pEZRThr567 in lung squamous carcinoma was significantly higher than that in the adja-cent and normal lung tissues (P<0.01).pEZRThr567 mainly localized on the cell membrane, and its over-expression signi-ficantly correlated with the differentiation, clinical stage and lymph node metastasis in lung squamous carcinoma.CON-CLUSION:pEZRThr567 may be an effective biomarker for prediction of malignant potential and poor prognosis of lung cancer.
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Objective To observe the clinical therapeutic effect of Capsule Long-Bi-Xiao(LBX) in treating benign prostatic hyperplasia(BPH).Methods 64 patients of deficiency of Qi and blood stasis type suffered with BPH were randomly divided into two groups.30 patients in the control group were treated with Capsule Long-Bi-Shu,while other 34 patients in the treated group were treated with LBX.Each group was observed for there months respectively.Results LBX decreased the international index of prostatic symptom(I-Pss) and the bother score(BS),improved the prostatic symptoms,raised the peak flow rate(Qmax) and the average flow rate(Qave),shrinked a portion of patients′ volume of prostate gland,reduced the volume of residual urine.After treatment,in the treated group,I-PSS and BS were reduced distinctly,and Qmax and Qave were heightened obviously,which was more effective than those in the control group.Conclusion LBX can achieve good action in the therapy of BPH by improving clinical symptoms,diminishing the volume of prostate and ameliorating the difficulty of urination.