Studies On Antitumor Effect Of DMDAI-L In Vitro And Its Mechanism

DMDAI-L (1,6-di-O-(methys-ulfonyl)-2,3,4,5-Dianhydro-L-iditol), an al-kylating cytostatic, was synthesized by using mannitol as material. According to the reports, it showed DMDAI-L was highly effective both on most of animal portability solid and ascitic tumours in vivo. As far as we know, research on the antitumor activity of DMDAI-L in vitro has never been reported. In this paper, we investigated the antitumor effect of DMDAI-L on a variety of human tu-mor cells in vitro for preliminary screening and its mechanism.Objective1. To investigate the antitumor effect of DMDAI-L on a varity of human tumor cells in vitro for preliminary screening.2. To evaluate cytotoxicity of DMDAI-L on human normal liver cells and human embryonic kidney cells.3. To study the apoptosis on HL-60cells which inducted by DMDAI-L, and its mechanism. Methods1. The proliferation inhibitory effect of DMDAI-L in different concentra-tions on a variety of human tumor cells was detected by CCK-8assay in vitro at different time.2. The cytotoxicity of DMDAI-L on human normal liver cells HL-7702and human embryonic kidney cells293were detected by CCK-8assay in vitro.3. Compared the proliferation inhibitory effects on HL-60cells among DMDAI-L and five clinical treatments of leukemia drugs, which in-cluding CDDP, ATRA, ARAC, DNR and CTX.4. The morphological changes of HL-60cells before and after administra-tion were observed through an inverted microscope, apoptotic bodies were observed on the fluorescence microscopy by the Hoechst33258staining assay, and the internal structures of the apoptotic cells were observed by transmission electron microscopy.5. DNA content for cycle changes of HL-60cells was detected by PI staining assay in flow cytometry, the apoptotic cells were analyzed by FCM using Annexin V FITC-PI double staining method.6. The changes of caspase-3activities in HL-60cells were measured by enzymatic visible substrate DEVD-pNA assay.7. The fluorescence changes of mitochondrial membrane potential (Δψm in HL-60cells were observed on the fluorescence microscopy by the the fluorescent probe JC-1marker method. Results1. The inhibition of proliferation of DMDAI-L on some kinds of human tumor cells, which came from human gastrointestinal, respirato-ry, reproductive system diseases, were not obvious, but there was highly effective on HL-60cells which was from blood system diseases. DMDAI-L could inhibit the proliferation of HL-60cells in a dose-time dependent manner, the IC5o values of HL-60cells for24h,48h,72h were57.514±2.089,5.234±0.754,2.451±0.092μg/mL2. DMDAI-L had very slightly inhibitory effect on human normal liver cells HL-7702and human embryonic kidney cells293in vitro. For example, the IC50value of human embryonic kidney cells293for72h was109.992±0.983μg/mL3. ATRA and CTX had slightly inhibitory effect on HL-60cells, while the IC50values of DMDAI-L, DNR, CDDP, ARAC on HL-60cells for48h were5.234±0.754,7.75±0.78,10.86±2.08,38.89±1.02μg/mL,4. Analysed by the Hoechst33258staining assay, there were condensation and marginalization exacerbated in the chromatin of HL-60cells after DMDAI-L treatment, it showed morphological changes associated with the character of apoptosis under fluorescence microscopy. Con-densation of chromatin at margins of nuclei, disintegration of nucleo-lus and vacuoles in cytoplasm were observed by transmission electron microscopy, indicating that DMDAI-L could induce apoptosis in HL-60cells.5. The apoptosis ratio of HL-60cells treated with DMDAI-L showed a clear upward trend in a dose-time dependent manner, and DMDAI-L could arrest cell cycle at S phase and G2/M phase (12h,24h and48h). 6. The caspase-3activity was significantly increased in HL-60cells after DMDAI-L treatment at a certain concentration and time range. DMDAI-L could significantly reduce the mitochondrial membrane potential in HL-60cells.Conclusion1. DMDAI-L has antitumor effect on human tumor cells in vitro with certain selectivity, especially, for human myeloid leukemia HL-60cells; DMDAI-L displays significant antitumor effect on HL-60cells.2. There is low toxicity for DMDAI-L on human normal liver cells HL-7702and human embryonic kidney cells293in vitro.3. Comparing to the five kinds of clinical anti-leukemia drug, there is more targeted and stronger proliferation inhibition on HL-60cells for DMDAI-L4. Studies on antitumor effect of DMDAI-L on HL-60cells show that its mechanisms are probably related to these factors as fellows:①DMDAI-L can arrest the cell cycle at S phase and G2/M phase, and induce the apoptosis of HL-60cells;②DMDAI-L can activate or regulate the caspase-3activity in HL-60cells at a cer-tain concentration and time range;③DMDAI-L can reduce the mito-chondrial membrane potential in HL-60cells.