Preliminary Study On Betulinic Acid And Betulin Derivatives' Anti-cancer Acitivity And Mechanism In Inducing Cell Apoptosis

Betulinic acid and betulin derivatives have selective cytotoxic function towards cancer cells and anti-HIV activity, with lower and better dosage limitation. In our study, six kinds of betulinic acid-like drugs: betulinic acid, 3,28-Di-O-succinyl betulin, 3,28-Di-O-glutaryl betulin, 3,28-di-O-Maleic-betulin, methyl ester of 3,28-Di-O-succinyl-glycylamino-betulin, N-[3-Oxo-20(29)-lupen-28-oyl]-glycine, are to be investigated by MTT method for their respective IC₅₀ values, representing in vitro ability to inhibit tumor growth, through which we find out sensitive cell SMMC7721 as a model for further evaluation of apoptosis inducing-related assays. LDH leakage activity and Trypan blue exclusion test are carried out to collaborate MTT test, confirmed the superior fatality rates and LDH leakage are discovered in the C28 modified derivatives of betulinic acid. On that basis, multiple methods, including morphology method, ROS (Reactive Oxygen Species) detection method, DNA Ladder AGE (agrose gel electrophoresis), monitor mitochondrial membrane potential and specific caspase acitivity assay (effector caspase-3/7 and initiation caspase-8, -9), all of these to present a preliminary evaluation on betulinic acid-like drugs'ability to induce tumor cell line SMMC7221 to apoptosis.1. In vitro MTT tests demonstrated that, betulinic acid-like drugs could inhibit cancer cells below: human hepatoma cell line SMMC7721, human gastric cancer cell line SGC7901, human breast carcinoma MCF-7, human nasopharyngeal carcinoma CNE, human tongue squamous carcinoma Tca8113, mice-derived melanoma cell B16. According to the IC₅₀ (50% inhibiting concentration) calculated, all five C28-modified betulinic acid-like drugs other than betulinic acid itself exhibit better growth inhibition efficiency towards carcinoma cell lines tested. Besides, statistical result from MTT shows no significant deviation within different drug dosage groups, while OD550nm measured drops side by side with drug concentration augmentation, which suggest both good cytotoxicity and selectivity of all betulinic acid-like derivatives tested. On the average, the activity order is :3,28-Di-O-succinyl betulin >3,28-Di-O-glutaryl betulin, 3, 28-di-O-Maleic-betulin, N-[3-Oxo-20(29)-lupen-28-oyl]-glycine > methyl ester of 3,28-Di-O-succinyl -glycylamino-betulin > betulinic acid。The most sensitive combination is 3,28-Di-O-succinyl betulin function towards SMMC7721 cell line, with corresponding IC₅₀ value about 1.39±0.55μmol/L.2. LDH(lactate dehydrogenase) leakage detection and trypan blue exclusion data suggest that all fatality rates and corresponding LDH leakage from six betulinic acid-like drugs is basically consistent with MTT result, fitting their property on tumor inhibition. The most active one, 3,28-Di-O-succinyl betulin, could drive SMMC7721 reach 90% above lethality rate within 24h at the concentration of 265.59μM, besides it can stimulate SMMC7721 cell membrane disrupted to release double times more than 80nM paclitexal does.3. Morphology characterization assays showed SMMC7721 how cell nuleus change under inverted fluorescence microscope. We find out with time pass by, cells begin to shrink and bubble, blue cell nucleus stained with Hoechst33342 become heavy dense and fragmented. The morphology observation from both betulinic acid-like drugs and paclitaxel as positive control present the same phenomenon, much different from uniformly light blue cell nucleus in negative control group. All the evidence suggest betulinic acid-like drugs we have in hand are able to induce tumor cell to apoptosis other than necrosis.4. In apoptotic DNA ladder extraction and 1.8% agarose gel electrophoresis test,"DNA Ladder"rather than smear is observed, representing hallmark of cell apoptosis in late stage. We further more confirm that C28-modified betulin- or betulinic acid- derivatives does induce tumor cells'nucleus to form apoptotic fragments more quickly with more strongly if under the same condition.5. Biochemically, highly distinctive caspase activity carries out with Caspase-3/7, -8, -9, the betulinic acid-like derivatives'ablity and capacity to induce SMMC7721 tumor cell to apoptosis is affirmative. Although caspase-3/7 derivative 3,28-Di-O-succinyl betulin and 3,28-Di-O-glutaryl betulin groups'caspase-3/7 activity is 50% ¹00% much more than positive control paclitaxel. On the other hand, drug which has disparity ratio of 8/9, 3,28-Di-O-succinyl betulin, is also the most outstanding betulin derivative tested. It may suggest that other apoptosis pathway is added up to the mitochondrial apoptosis mechanism, or the mitochondrial stimulation is accelerated and pushed forward much stronger than betulinic acid, unmodified group, that is to say, initiation caspase-8,-9 bring their peak arrive ahead of usual, then downstream effector caspase-3/7 has a dramatic increase afterwards. The latter one is more plausible when considering the time series consequences relationship plot, that 3,28-Di-O-succinyl betulin could bring caspase-8 and -9 reach the peak within 2h.6. As observed in ROS(Reactive Oxygen Species) assay, compared with betulinic acid, five C28-chemically modified betulin derivative could make tumor cell produce ROS more and fast, reach the climax within 2h, and become two times more than positive control (50μg/ml Rosup and 80nM paclitaxel), which is a very good reason to believe apoptosis is happened ahead of schedule and with much more magnitude.7. Futhermore, the MMP (mitochondrial membrane potential) descends gradually as time goes by. The decrease of MMP and rise of ROS production, as former references'presumptions and conclusions, are both prerequisite for betulinic acid to induce tumor cell to apoptosis. These data of mitochondrion also strongly suggest betulinic acid-like drugs tested is fitting the shape of apoptosis and even more.On the whole, gathering data or phenomenon from MTT, LDH and typan blue exclusion test for getting general picture, to apoptosis assay including morphology test, biochemical test, agarose gel eletrophoresis, or in vitro caspase activity assay, all the results demonstrated that betulinic acid-like drugs tested have superior apoptotic and tumor-inhibition activity, and five C28-chemically modified derivatives increase a lot on the tumor inhibition and apoptosis induction ability than original betulinic acid. Our research provides a good foundation for monitoring apoptotic parameters in betulinic aicd like drugs-functioned tumor cell, and is also important reference for further research on tumor apoptosis pathway and mechanism.