Establishment Of LNCaP Xenograft Prostate Cancer Model In Balb/C Mice

Objective:(1)To explore LNCaP cells undergo apoptosis and the mechanisms of apoptosis in response to different oxygen level.(2)To explore the take rate of LNCaP subcutaneous xenograft model under different oxygen level.(3)To explore the establishment methods of LNCaP orthotopic prostate cancer model.Materials and methods:(1)Cell viability were assayed by CCK-8 assays.The production of ROS was measured by a fluorescent probe assay.Apoptosis,mitochondrial membrane potential(??m),and the cell cycle of LNCaP cells were analyzed by flow cytometry.Western blot analysis detect protein expression levels of HIF-1? and MAPK pathway.(2)LNCaP subcutaneous xenograft model divided into groups according to the cell lines,Matrix glue and air composition.The initial tumor formation time and take rate were observed.(3)Anatomical structure of prostate lobes in Balb/c mice was analyed by stereoscopic microscope.Testosterone propionate was administrated to prompt prostate enlargement.Orthotopic nude mouse model of LNCaP human prostate cancer was established under stereoscopic microscope.Prostate tumor growth in an orthotopic mouse model was monitored using ultrasound.Results:(1)The viability of LNCaP cells were markedly suppressed under acute hypoxia.Acute hypoxia exposure for 24 h induced significant ROS production.LNCaP cells exhibited a time-dependent increase in apoptosis under acute hypoxia by flow cytometric analysis.Furthermore,acute hypoxia induced the collapse of the ??m of LNCaP cells,and it also increased cell cycle arrest at the G0/G1 phases in a time-dependent manner.Acute hypoxia enhanced the activation of HIF-1? and cleaved caspase 3,activated MAPK pathway of ERK and p38 MAPK protein in LNCaP cells.(2)The initial formation time of PC-3+Matrix+air group was not statistically different from that of PC-3 +Matrix group,while the take rate of LNCaP+Matrix+air group was higher than that of LNCaP+ Matrix group.(3)Male Balb/c nude mice had a smaller spatial range in the ventral ventral lobe,while the dorsal lobe was triangular pyramid with the spatial range was relatively large.The operative time of the testosterone propionate group was shorter than that of the control group in the Balb/c nude mice orthotopic xenograft model.Ultrasound can monitor the initial tumor formation time and tumor volume changes.Prostate orthotopic xenograft tumor growth in the prostate glandular interstitial.Androgen receptor(AR)and prostate specific antigen(PSA)expression was positive.Conclusion:(1)Acute hypoxia induces substantial apoptosis in LNCaP cells,and activates MAPK apoptosis pathway.(2)Subcutaneous injection with the addition of oxygen in the establishment of LNCaP subcutaneous orthotopic model is a simple and convenient method to improve the take rate of tumor.(3)The orthotopic xenograft Balb/c nude mice model of LNCaP cells is constructed successfully under stereoscopic microscope.Ultrasound is a noninvasive method of examination that can be used for the monitoring of orthotopic tumors growth.Passage one The study of prostate cancer LNCaP cells under acute hypoxia in vitro Part one Basical functions in response to acute hypoxia of prostate cancer LNCaP cellsObjective: To assess serum-deprived LNCaP cells viability,morphological changes,and migration.Materials and methods: Serum-deprived LNCaP cells were cultured and exposed to either normoxic(21.0% O2)or hypoxic(1.0% O2)conditions.Morphological changes in nuclei were evaluated with Hoechst 33258 staining under fluorescence microscopy.Cell viability and migration were assayed by Cell Counting Kit-8 and scratch assays,respectively.Results: Under acute hypoxia,the inhibition rates of LNCaP cells were 42.24 ± 4.72% and 78.34 ± 8.44% at 24 h and 48 h,which were significantly higher than those of LNCaP cells under normoxia(32.02 ± 3.65%,P<0.05;35.12 ± 5.44%,P<0.01;respectively).Under normoxic conditions,a subset of LNCaP cells showed a neuroendocrine-like cells at 48 h.While under acute hypoxia,the LNCaP cells tended to aggregate.Nuclear morphological analysis with Hoechst-33258 staining showed that apoptotic cells could be observed under both normoxia as well as acute hypoxia.LNCaP cells had a decreased capacity for migration in the presence of acute hypoxia.Conclusion: LNCaP cells tended to form multicellular aggregates,and the viability and migration of LNCaP cells were markedly suppressed under acute hypoxia.Part two Study of mitochondrial function under acute hypoxia in LNCaP cellsObjective: To detect the change of mitochondrial membrane potential(??m)and ROS generation in LNCaP cells under acute hypoxia.Materials and methods: The change of mitochondrial ??m was determined using the JC-1 Mitochondrial Membrane Potential assay kit.Intracellular accumulation of ROS was measured by DCFH-DA fluorescent probe assay.Results: The percentage of cells with JC-1 green fluorescence was not significantly altered under acute hypoxia at 12 h(7.22 ± 1.46%),compared with that under normoxia(4.90 ± 1.22%,P>0.05).However,significant increases in the percentage of LNCaP cells with JC-1 green fluorescence were observed under acute hypoxia at 24 h(27.91 ± 5.34%)and 48 h(36.62 ± 6.01%).LNCaP cells produced significantly more ROS under acute hypoxia at 24 h(32,492.00 ± 10,344.55 AU vs.11,300.66 ± 3969.75 AU at 12 h,P<0.01).The MFI rapidly reached a peak at 24 h under acute hypoxia,compared with that under normoxic conditions at 24 h(32,492.00 ± 10,344.55 AU vs.11,899.11 ± 889.96 AU,P < 0.01).Conclusion: Acute hypoxia lead to an decrease in mitochondrial membrane potential and increase in ROS production.Part three Mechanisms of apoptosis in response to acute hypoxia of LNCaP cellsObjective: To analyze mechanisms of LNCaP cells apoptosis under acute hypoxia.Materials and methods: Cells were stained with an Annexin-V/PI Apoptosis Detection Kit to determine early apoptosis or late apoptosis/death of cells.PI staining was used to assess stages of the cell cycle.HIF-1?,caspase 3,and MAPK signaling in p38,ERK,and JNK was examined with western blot analysis.Results: The percentages of early apoptotic cells under acute hypoxia at 24 h,and 48 h were 15.32 ± 2.34% and 24.11 ± 4.70%,respectively,which were significantly greater than those under normoxia at the same time points(24 h: 4.31 ± 0.92%,P<0.01;48 h: 6.22 ± 1.12%,P<0.01).The percentages of late apoptotic cells under acute hypoxia at 12 h,24 h,and 48 h were 15.84 ± 2.81%,26.81 ± 5.52%,and 46.42 ± 8.43%,respectively,showing a significant increase at 48 h compared with that under normoxia(48 h: 22.94 ± 4.14%,P<0.05).The LNCaP cell populations in the G0/G1 phases under acute hypoxia at 48 h were significantly increased compared to those of LNCaP cells under normoxia(53.36 ± 6.85% vs.70.75 ± 9.39%,P<0.01).HIF-1? and caspase 3 induced by acute hypoxia.Acute hypoxia active ERK and p38 MAPK kinase,and anti?active JNK MAPK kinase.Conclusion: Acute hypoxia exacerbated cellular apoptosis and induced increased G0/G1 arrest in LNCaP cells in a time-dependent manner.The pro-apoptotic activity of acute hypoxia in LNCaP cells may induce by both ERK and p38 pathways.Passage Two Establishment of subcutaneous LNCaP human prostate cancer model in nude micePart one Methodological study on establishment of subcutaneous xenograft model of LNCaP cells in nude miceObjective: To explore the establishment of subcutaneous xenograft model of LNCaP cells in nude mice.Materials and methods: Nude mice were divided into 8 groups according to the different components of injected cells,air,and Matrix.The initial tumor formation time and tumor growth rate were monitored.Results: There were no significant difference in the initial tumor formation time between PC-3+matrix and PC-3+air+Matrix group(P>0.05).The initial tumor formation time were longer than in LNCaP+Matrix group than that of LNCaP + Matrix+air group(P<0.05).The take rates of LNCaP+air+Matrix group were highter than that of LNCaP+Matrix group(P<0.05).Conclusion: Matrix glue is a necessary condition for LNCaP to establish subcutaneous xenografts.On this basis,the addition of air is beneficial to improve the take rate of LNCaP xenografts nude mice.Part two Effects of different oxygen concentrations on the proliferation rate of LNCaP cells in nude mice under subcutaneous xenograftObjective: To investigate the effect of different oxygen level on tumor take rate of LNCaP subcutaneous xenograft model in nude mice.Materials and methods: According to the injection of gas oxygen level,male mice was divided into three groups.LNCaP+Matrix+air group was administered LNCaP,Matrix,and air.LNCaP+Matrix+40% oxygen group was administered LNCaP,Matrix,and 40% oxygen.LNCaP+Matrix+40% oxygen group was administered LNCaP,Matrix,and 60% oxygen.The three groups were observed for 90 days and the take rate between the groups were compared.Results: The take rates of LNCaP+Matrix+air group,LNCaP+Matrix+40% oxygen group,and LNCaP+Matrix+60% oxygen group were 36.36%,41.67% and 25.00%,respectively.There were no significant difference in take rate between the three groups(P<0.05),but higher than those without administration of oxygen in LNCaP group and LNCaP + Matrix group.Conclusion: Injection of LNCaP cell suspension subcutaneously with administration of oxygen can increase the take rate of LNCaP nude mice subcutaneous xenograft.Passage Three Establishment of orthotopic LNCaP model of prostate cancer in Balb/c mouse Part one Microanatomical study of Balb/c mouse prostateObjective: To study the microscopic anatomical characteristics of proatate in male Balb/c nude mice.Materials and methods: 10 male 8-week Balb/c nude mice were observed withstereo microscope 6.5 times in 4 section to study the mice prostate.Results: Various morphologic features of the mice prostate complex were characterized by anatomic dissection and by stereo microscope.The anterior lobe was the largest lobe in four lobes in proatate of Balb/c nude mice,followed the dorsal lobe,while the ventral and lateral lobe was relatively small.Conclusion: Under the stereo microscope can distinguish the four lobes of prostate in Balb/c nude mice.Part two Histological analysis of the Balb/c mouse prostateObjective: To study the histological features of the prostate lobe of Balb/c nude mice.Materials and methods: Balb/c nude mice were examined by hematoxylin and eosin(H&E)staining,and the histological features of the gland were observed under microscope.Results: Balb/c nude mice have different histological features,in which the peritoneal epithelium cells of the ventral glands was thicker.Capsule incontinuity was seen in ventral prostate,and prostatic intraepithelial neoplasia was seen in dorsal prostate.Conclusion: Balb/c nude mice with Capsule incontinuity in ventral prostate may be susceptible to exogenous stimulation.Dorsal prostate in nude mice is ideal for studying prostate cancer.Part three Establishment of LNCaP prostate cancer orthotopic nude mice modelObjective: To investigate the establishment of LNCaP nude mice orthotopic xenograft and the ultrasonographic monitoring of xenograft tumor.Materials and methods: The LNCaP orthotopic xenograft model with or without testosterone propionate group(control group)was established.Tumor growth was monitored by ultrasound.LNCaP nude mice orthotopic tumor was stained with HE and AR,and PSA immunohistochemical analysisResults: The median operative time was 12 min in the testosterone propionate group and 17.5 min in the control group.The operation time of the testosterone propionate group was significantly shorter than that of the control group(P<0.01).The take rate of control group and testosterone propionate group was 37.50%(3/8)and 22.22%(2/9),respectively.There were no significant difference between the two groups(P = 0.49).There were no significant difference in the growth rate of xenograft tumor monitored by ultrasonography(P=0.54).Large hemorrhagic necrotic area can be seen inside the orthotopic tumor.Tumor capsule was thick.Tumor growth in the prostate stroma,the surrounding glands were significantly compressed.Local metastases were commonly seen.AR and PSA immunohistochemical staining was positive.Conclusion: Administration of testosterone propionate can reduce the operation time in the establishment of orthotopic model of LNCaP prostate cancer.The application of ultrasound is a useful imaging modality in monitoring tumor growth in an orthotopic LNCaP mouse model.