Studies On Apoptotic Mechanism Of MDCC-MSB1 Induced By LaCl₃ In Vitro

Some early studies imply that high level of rare earth has some hereditary toxieity,while low level rare earth may be very effeetive in cancer therapy.As the research of rare earth goes further every day in the regions of medieine and bioehemistry,it will play a more important role in the life seienee. MD may be take as a modle of cancer desease research by virtue of resemblance with mammal cancer. In the study we will focus on inhibitory action and LaCl₃-induced apoptotic pathways with an emphasis on mechanisms in LaCl₃-induced apoptosis including its effect on Using flow cytometry, TUNEL, electrophoretic analysis of DNA, fluorescent staining, electronmicroscope etc., studied the changes of apoptotic morphology and biochemistry as well as possible regulative mechanism by detecting intra-cellular Ca²⁺ concentration([Ca²⁺]i), chromosome DNA damage, cell cycle, mitochondria membrane potentia(lΔψm),Caspase-3,Caspase-9 activity and CaM,Caspase-3 mRNA expression level. in MDCC-MSB1. The mechanism will provide evidence in the treatment of MD and other cancer diseases. The results showed as follows:1. Determine the activation of MDCC-MSB1 cell and the level of LDH by MTT assay and dinitro-phenylhydrazine(DPNH) chromometry. The change of cell cycle and CaM mRNA expression were detected respectively by RT-PCR and flow cytometry. The result show that cell proliferation may be inhibited by LaCl₃ and inhibition ratio exhibite concentration dependent. The result show that LaCl₃ induced MDCC MSB1 apoptiosis by G0/G1 cycle inhibition of the cells.2. Cell growth condition was detected by inverted microscope, Morphology changes were detected by AO/EB double fluorescence coloration, HE staining, transmission electron microscope, phosphatidylserine(PS)was detected by flow cytometry, which revealed that MDCC-MSB1 apoptosis was induced by LaCl₃. Apoptosis ratio exhibite concentration dependent.3. Applying agarose gel electrophoresis, alkality SCGE, TUNEL detected MDCC-MSB1 DNA damage, it showed that LaCl₃ induced MDCC-MSB1 apoptosis by increasing [Ca²⁺]i, activiting endogenous endonuclease, breaking DNA in nulceosome.4. The result of detection on anti-oxidative function dedicated that LaCl₃ lead to the increase of MDCC-MSB1 NOS activity and NO content; the derease of anti-oxidative function, which lead to the oxidative stress. Possibly, this was one of the mechanisms of apoptosis induced by LaCl₃. 5. The changes of,Δψm, Caspase-3, Caspase-9 activity and Caspase-3 mRNA expression were detected by Fura-2/AM fluorometric method, flow cytometry, Colorimetric Assay Kit and semiquantitative PCR, respectively. The research showed thatΔψm was encreased, [Ca²⁺]i, Caspase-3, Caspase-9 activity and Caspase-3mRNA expression were increased, it revealed that LaCl₃ induced MDCC-MSB1 apoptosis by activating Caspase access.LaCl₃ may inhibit MDCC-MSB1 growth and induce apoptosis. The results indieate that the higher eoneentration of rare earth can inhibit the cells growth,whereas the lower coneentration of rare earth has no influence on eell growth, moreover,the effect became stronger as the concentration increased.