Studies On Anti-tumor Activities Of MS-275-6 And Its Mechanism Of Action

Histone deacetylase inhibitors are a series compounds, can inhibit cell proliferation, induce cell cycle arrest and stimulate apoptosis of cancer cells, including trichostatin A TSA, the hydroxamic acids, suberoylanilide hydroxamic acid (SAHA), butyric acid BA, benzamides derivative,such as MS-275.Objective: Our laboratory obtains a series of novel structures derivatives through the the structure transformation to the benzoyl ammonia class derivative MS-275, in which MS-275-6 has the stronger anti-tumor activity in vitro,also has the good quantity-effect relations. This paper has further studied the inhibitory action and characteristic of MS-275-6 to tumor cells and animal transplant tumor raise in vitro,and initially has discussed its function mechanism.Methods:1 The Growth inhibition of a combination of MS-275-6 on tumor cells and nomor cells was analyzed by cell viability using MTT and SRB assay.Inoculate tumor cells and human kidney proximal tubular epithelial cell (HKC) in exponential phase of growth by using 96-hole cultivation plate , adding MS-275-6(10μmol/L,1μmol/L,0.1μmol/L,0.01μmol/L) after 24 hours of cultivating. Measure the growth inhibiting rate and GI50 of MS-275-6 in vitro towards tumors cell by MTT and SRB method after 96 hours of cultivating.2 Microscopic structure changes was observed by inverted microscope and nuclear morphological assessment of apoptotic cells and dead cells were observed by fluorescence microscopy.We assessed the morphological change of effects MS-275-6 on K562 cells by Inverted Microscope (×200).And exposed Hela cells with MS-275-6 for different time, adding Hoechst33342, observed and photographed it on fluorescence microscope to reserve the result.3 We transplant entity-type tumor carneus S180 into Kunming mouse's body , after 24 hours of transplanting, adding MS-275-6 (40mg/kg/d and 80 mg/kg/d)successively in ten days,then killing these mousse ,weighing them and the fleshy tumor,finally observing the therapeutical effect of MS-275-6 towards entity-type tumor carneus S180.4 The DNA ladder was revealed by agarose gel electrophoresis. We extract DNA from the Hela cells, treated with MS-275-6 for 48h, for agarose gel electrophoresis. and paclitaxel (0.1μmol/L, 18 h) as a positive control.5 The cell cycle state and apoptotic rate were examined by flow cytometry analysis (FCM). We collect Hela cells in exponential phase of growth, adding MS-275-6(2μmol/L,4μmol/L), then keeping on cultivating the Hela for different time(24h,36h,48h,72h) and detected by flow cytometry by turns. 6 The mRNA expression of p53,bcl-2,bax,p21 in Hela cells was semi-quantified by reverse transcription PCR(RT-PCR).7 The expression of CDK2 in Hela cells was semi-quantified by Western-blot.Results:1 Effects of MS-275-6 on the proliferation of cancer cellsMS-275-6 selectively inhibited growth of cancer cell lines at submicromolar levels after 48~72 h of exposure, whereas higher concentrations and longer exposure times were required to retard the growth of normal dermal human fibroblasts. In a series of experiments, eight tumorigenic human cells, including human osteosarcoma cell (HOS), human lung adenocacinoma cell (A549), human cervical cancer cell (Hela), human ovarian carcinoma cell (SKOV3), human erythroleukaemia K562 cells (K562), human colon cancer cells(HT29), human breast cancer cells (MCF-7) and human stomach carcinoma cell (BGC-823) were chosen to determine the cytotoxic activity of MS-275-6, and one normal human kidney proximal tubular epithelial cell (HKC) were chosen to determine the cytotoxic activity of MS-275-6. After incubating for 96h, increasing concentrations of MS-275-6 (1.0～10.0μmol/L) led to a gradual decrease of viable cells fraction. MTT assay showed that IC50 was 4.01μmol/L (BGC-823) to 8.80μmol/L (K562). Because of these variations, the cytotoxic activity of MS-275-6 had been determined against a panel of human tumor cells of different origins. Results of SRB assay showed anticancer activity by the cytostatic effect when it was incubated in the low concentration, the GI50 of MS-275-6 to Hela, BGC-823 was 6.49μmol/L, 4.51μmol/L respectively, but MS-275-6 showed anti-cancer effect through the cytotoxic effect when it was incubated in the high concentration.2 Inhibition of tumor growth in vivo by MS-275-6The effect of MS-275-6 on tumor growth was studied in KunMing mice using transplanted models with sarcoma S180. Intragastric administration of MS-275-6 (40mg/kg and 80mg/kg) was performed every day, followed with tumor cells inoculation. Mouse S180 sarcoma was sensitive to MS-275-6,40mg/kg and 80mg/kg MS-275-6 treatment resulted in 39.3% and 36.0% (P<0.05) inhibition of tumor growth compared with untreated control. Neither death nor altered weight modification of the host occurred, MS-275-6 seemed little toxicity in our experimental conditions. Effects of MS-275-6 in vivo showed that daily lavage of MS-275-6 at less than toxic doses reduced the tumor weights.3 Causing cell cycle arrest by MS-275-6Flow cytometry studies revealed that tumor a cells arrested in the G1 phase of the cell cycle after compound treatment. After treatment with 2.0~4.0μmol/L of MS-275-6 for 36h, the number of cells in G1 phase was higher than that in untreated cells. The percentage of cells arrested in G1 phase increased with the increasing of concentration.4 Inducing apoptosis by MS-275-6 MS-275-6 induced morphological changes by Giemsa and Hoechest staining, which were characteristics of apoptosis. Contrast cells displayed excellent growth characteristics -polygonal shape with round large nucleus featuring prominent multiple nucleoli, and well spread on the growth surface. MS-275-6 evoked typical apoptotic features such as membrane blebbing, cell shrinkage and detachment, and nuchelar condensation and fragmentation.Flow cytometric analysis of Hela cells exposed to MS-275-6 confirmed the morphological observations above. An increased sub-G1 population at 48 h (reminiscent of apoptotic cells) was observed in the cancer cell line. The DNA fluorescence histograms of PI-stained cells showed the low DNA stainability of the MS-275-6-treated apoptotic cells, which resulted in a distinct, quantifiable region below the G1 peak. In contrast, the G1 peak predominated in control cells. Quantification of dose-dependency was done by monitoring the amount of nuclei with subdiploid DNA content with flow cytometry. The apoptotic Hela cells increased up to 31.2% , 72.7% after incubated in MS-275-6 for 48 h. The effect was also time-dependent. The proportions of apoptotic Hela cells incubated in MS-275-6 for 48h were higher than in the same concentration for 36h. The dead cells were increasing with the prolonged time and the enhancive concentration, so the apoptotic cells decreased relatively. Finally, agarose gel electrophoresis showed typical DNA fragmentation pattern and confirmed the apoptosis induced by MS-275-6.DNA fragmentation caused by MS-275-6 was dose-dependent. The intensity of DNA fragments increased as increasing amounts of MS-275-6 (2.0~8.0μmol/L) was added to the cells. As a positive control, cells were treated with paclitaxel (0.1μmol/L, 18 h).5 Regulating expression of P53 mRNA , Bcl-2 mRNA, Bax mRNA and p21 mRNA by MS-275-6.Expression of P53 mRNA , Bcl-2 mRNA, bax mRNA and p21 mRNA in Hela cells exposed to MS-275-6 was investigated by RT-PCR. Data showed that P53 mRNA, Bax mRNA and p21 mRNA mRNA levels increased,meanwhile the expression of Bcl-2 mRNA decreased for 24h culture in the present of MS-275-6.6 Western blotting experiments showed apoptosis induced by MS-275-6 was associated with the an decreased of CDK2.Conclusions: MS-275-6 inhibited partially purified human HDA and caused hyperacetylation of nuclear histones in various tumor cell lines. It behaved in a manner similar to other HDA inhibitors, such as benzamides derivative,such as MS-275; MS-275-6 induced p21 and and changed the cell cycle distribution, decrease of S-phase cells, and increase of G1-phase cells. The in vitro sensitivity spectrum of MS-275-6 against various human tumor cell lines showed a pattern different than that of a commonly used antitumor agent. MS-275-6 showed obvious anticancer activity by inducing apoptosis and causing cell cycle arrest in the dose-dependent manner, which were accompanied by upregulating P53, p21 and bax, downregulating Bcl-2.These results suggest that MS-27-275 acts as an antitumor agent through HDA inhibition and may provide a novel chemotherapeutic strategy for cancers insensitive to traditional antitumor agents.