ABSTRACT
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.
ABSTRACT
BACKGROUND AND OBJECTIVES: It is well known that Prostaglandin E2 (PGE2) is the most predominant prostaglandin in squamous cell carcinoma and that PGE2 synthesis is suppressed by retinoid. The purpose of this study was to confirm whether (N-4-Hydroxyphenyl) retinamide (N-4-HPR) suppressed PGE2 synthesis, and investigate its inhibitory mechanism on PGE2 synthesis in squamous cell carcinoma. MATERIALS AND METHOD: MDA 886Ln was used as the squamous cell carcinoma cell line. We evaluated the effects of four retinoids (all-trans-RA, 13-cis-RA, retinyl acetate, and N-4-HPR) on PGE2 synthesis: the effect of N-4-HPR concentration on PGE2 synthesis and Cox-2 mRNA, the effect of N-4-HPR on Cox-2 protein, and the effect of N-4-HPR on the cyclooxygenase activity. RESULTS: Among the four retinoids, N-4-HPR was the most potent suppressor of PGE2 synthesis. N-4-HPR suppressed PGE2 synthesis, but N-4-HPR did not suppress Cox-2 mRNA or Cox-2 protein. Cyclooxygenase activity was suppressed by N-4-HPR. CONCLUSION: With these results, we suggest that the inhibitory mechanism of N-4-HPR on the PGE2 synthesis may be suppression of the cyclooxygenase activity, and Cox-2 mRNA and protein were not suppressed by N-4-HPR.
Subject(s)
Carcinoma, Squamous Cell , Cell Line , Dinoprostone , Prostaglandin-Endoperoxide Synthases , Retinoids , RNA, MessengerABSTRACT
The retinoic acid, one of the most popular agents for chemoprevention can inhibit the proliferation of many cancer cells including neuroblastoma and glioblastoma but there is increasing demand reaccessing its in vitro inhibitory effect on the tumor proliferation because of poor responsiveness from recent clinical trial for malignant brain tumor with retinoic acid. If was known to effect on tumor cells by diffferentiation and apoptosis so that its effect was expected greater in pediatric brain tumor than in adult brain tumor, but there is no report on the effect of retinoic acid in medulloblastoma cell proliferation except all-trans and 13-cis retinoic acid. Therefore, we compared the effects of all-trans, 13-cis, and 9-cis retinoic acid and N-(4-hydroxyphenyl) retinamide to inhibit proliferation of medulloblastoma tumor cells. Medulloblastoma cells were derived from primary culture of patient's specimen. We estimated the rate of growth inhibition of each tumor cells using MTT assay in the concentration from 10-12 M to 10-5 M of all-trans, 13-cis, and 9-cis retinoic acid and N-(4-hydroxyphenyl) retinamide. Medulloblastoma cells showed more than 30% growth inhibition by all-trans, 12% by 13-cis, 20% by 9-cis retinoic acid and 7% by N-(4-hydroxyphenyl) retinamide at 14 days culture on the concentration of 10-6M. In conclusion, significanty and dramatic effect by, especially, all-trans retinoic acid, moderate response by 13-cis retinoic acid and variable or poor response by 9-cis retinoic acid and N-(4-hydroxyphenyl) retinamide.