Local Anesthetic Bupivacaine Induced Ovarian And Prostate Cancer Apoptotic Cell Death And Underlying Mechanisms In Vitro

Part Ⅰ The effects of bupivacaine on cancer cell viability and chemotherapy sensitivityObjective:To investigate the inhibition effects of bupivacaine on cancer cell viability, and if it can modulate the sensitivity of cancer cells to chemotherapy. What’s more, to clarify if the apoptotic effects of bupivacaine via the modulation of caspase activity.Methods:Human ovarian carcinoma (SKOV-3), prostate carcinoma (PC-3) and human proximal tubular cell (HK-2) were cultured in vitro. All the cells were seeded in24wells plate and waited for24h to reach70%-80%confluence before each experiment. Cells of experimental groups were cultured with bupivacaine at concentrations ranging from1μM to1mM and the same volume normal saline was applied in control group. After24or72h, cell viability was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. For assessing the different effects of bupivacaine on cancer cell and normal cell, HK-2cells were treated with1mM bupivacaine for24h, then use MTT assay to measure cell viability. Other cohort cultures of SKOV-3and PC-3cells were treated with1mM bupivacaine or together with100nM chemotherapy drug taxol, and cell viability was evaluated via MTT assay as well after24h. In caspase assessment groups,1mM bupivacaine was added for24h. The expression of caspase3,8and9was detected by immuno staining. Caspase inhibitor and FAS ligand neutralization antibody was applied accordingly to clarify the involvement of apoptotic pathway and death receptor.Results:MTT showed that only highest concentration (1mM) of bupivacaine suppressed viability of SKOV-3and PC-3cells after24and72h treatment. Cell viability of both cell lines were reduced after100μM bupivacaine treatment, but it didn’t reach statistically difference. In both SKOV-3and PC-3cell lines, the decrease of cell viability was more evident after72h bupivacaine treatment when compared with24h group, which indicates that the killing effects of bupivacaine to cancer cell is time dependent. More viability descent was found in SKOV-3cell line than PC-3, which indicates the diverse killing effects of local anesthetic bupivacaine to cancer cells. Although slightly decreased, no statistically difference was found in HK-2cell line after24h bupivacaine treatment, which suggests us that cancer cells are more vulnerable to bupivacaine than normal cells. At last, the results showed additive effects rather than synergetic effects after24h treatment of100μM or1mM bupivacaine treatment together with taxol.The analysis of immunofluorescence indicated that after the treatment of1mM bupivacaine for24h, the expression of caspase3,8and9were all elevated in SKOV-3cells, and caspase8and9inhibitor partly reversed bupivacaine induced cell death. The similar effects of caspase3and9were also found in PC-3cells, but not of capase8. The subsequently administration of caspase8inhibitor faild to reverse bupivacaine induced cell death, but caspase9inhibitor did. At last, no significantly change was found on the use of FAS ligand neutralization antibody.Conclusion:Bupivacaine treatment induced dose and time dependent cancer cell death. This effects was associated with the activation of apoptotic pathway, and irrelevant to the death receptor. Part Ⅱ The effects of bupivacaine on cancer cell proliferation and migrationObjective:To investigate the inhibition effects of bupivacaine on cancer cell proliferation and migration.Methods:Human ovarian carcinoma (SKOV-3), prostate carcinoma (PC-3) were cultured in vitro. All the cells were seeded in24wells plate and waited for24h to reach70%-80%confluence before each experiment. In cell proliferation experiments,1mM bupivacaine was added into the culture medium of SKOV-3and PC-3,24hours later, the expression of essential proliferative marker Ki67was evaluated via immunostainning assay. In cell migration experiments, we applied scratch assay. Cancer cells were seeded in petri dishes and to form a confluent monolayer at the bottom after24h. Then, a cross wound was scratched on this monolayer by1ml pipet tip. Before taking each image, a mark was made at the bottom of the dish to make sure that all the images were taken at the same site. After another24hours incubation with or without1mM bupivacaine, the second image was taken at the same site. The wound healing status was compared by Image-Pro Plus software based on these images. The results were presented as (initial wound area-remaining wound area)/(initial wound area) to evaluate the migration rate of the cells.Results:The assessment of immunofluorescence strength showed that, in both cancer cell lines, the expression of Ki67was dramatically decreased after 24h treatment of1mM bupivacaine. This result suggests1mM bupivacaine can effectively inhibit cell proliferation. For cell migration assay, wound healing rate was apparently reduced in both cell lines after24h bupivacaine treatment, which indicates that bupivacaine dampens migration ability of cancer cell.Conclusion:Bupivacaine treatment decreased cancer cell proliferation and migration ability. Part Ⅲ The role of GSK-3β in bupivacaine induced cancer cell death Objective:To investigate the activation of GSK-3p in bupivacaine induced SKOV-3and PC-3cell deathMethods:Human ovarian carcinoma (SKOV-3), prostate carcinoma (PC-3) were cultured in vitro. All the cells were seeded in24wells plate and waited for24h to reach70%-80%confluence before each experiment. After24h incubation with1mM bupivacaine, phosphorylated total GSK-3β, GSK-3βtyr216(active form) and GSK-3βser9(inactive form) were assessed by immnunostaining assay. In other cohort studies, along with the administration of lmM bupivacaine, GSK-3inhibitor SB-216763was used to inhibit GSK-3β. What’s more, other cells were cultured with siRNA targeting GSK-3p or unspecific scramble siRNA for6h, then lmM bupivacaine was superposed for24h incubation. At last, the expression of cleaved caspase3, caspase8and caspase9was assessed by immunostaining and the final survival situation of cancer cells were also evaluated.Results:After1mM bupivacaine treatment for24h, the amount of phosphorylated total GSK-3β, GSK-3βtyr216and GSK-3βser9were all increased in SKOV-3cell line. Specially, the amount of active form GSK-3βtyr216was increased one time after the treatment. In another hand, phosphorylated total GSK-3β, GSK-3βtyr216and GSK-3βser9were equal to control group after treatment in PC-3cell line.Conclusion:Bupivacaine treatment increased phosphorylation level of total GSK-3β, GSK-3βtyr216and GSK-3βser9in SKOV-3cells. Among them, the elevation of GSK-3βtyr216was most evident. But the same change could not be found in PC-3cell line. Both inhibitor and siRNA treatment partially reduced bupivacaine induced SKOV-3cell death. But these reagents did not have any effect in PC-3cells. Following GSK gene knockdown, we found that the expression of GSK-3β was very low, and caspase3,8,9cleavage and pGSK-3βtyr216activity was decreased in SKOV-3cells via immunostaining assessment.