Therapeutic Effect Of4-Amino-2-Trifluoromethyl-Phenyl Retinate On Nude Mice Transplanted With Human Gastric Cancer SGC-7901Cells And The Possible Mechanism

Since all-trans retinoic acid (ATRA) differentiation therapy of acutepromyelocytic leukemia successfully, ATRA had become the hotspot of anti-cancerresearch as the representative of differentiation inducing agent. Side-effects of ATRAsuch as retinoic acid syndrome limited its further application in the clinical. So thatthe new, efficient, low toxicity of differentiation inducing agents became researchdirection. Our laboratory synthesized a series of retinoids derivatives using ATRA asraw materials. After screening of the activity, the pharmacological activity of4-amino-2-trifluoromethyl-phenyl retinate (ATPR) was higher than others. It has significanteffect of suppression proliferation and inducing differentiation on leukemia cells.Pharmacodynamic studies of the solid tumor cell in vitro found that ATPR hasinducing differentiation effects on a variety of gastrointestinal tumor. In this study, weestablished the model of human gastric cancer cell SGC-7901xenografted in nudemice to explore the effect of ATPR on gastric cancer cells in vivo and provideexperimental basis for its clinical application. Silencing of SGC-7901cell RARβgene by the RNA interference and then using ATPR, observing the influence ofproliferation differentiation capacity of ATPR on SGC-7901cell in vitro, aims toclarify the mechanism of ATPR anti-proliferation and iducing differentiation inSGC-7901.Objective: To investigate the anti-tumor effect of4-amino-2-trifluoromethyl-phenylretinate (ATPR) on nude mice implanted human gastric cancer cell line SGC-7901 and silencing of SGC-7901cell RARβ gene by the RNA interference and then usingATPR, observing the influence of proliferation differentiation capacity of ATPR onSGC-7901cell in vitro.Methods:1Vaccination:10male nude mice were feeded for1weeks under the condition ofSPF. The logarithmic growth phase cells were collected by trypsin digestion and everymouse was inoculated with2×106SGC-7901cells. When the tumors grow to about1cm3, break the neck of mice and injecte suspension of tumor tissue subcutaneouslyinto nude mouse armpit under aseptic conditions. Three generations later, tumor tissueas a source of subcutaneous transplantation tumor were injected into103male nudemice armpit.2Groups and administration methods: after a week inoculation, only91mice wererandomly divided into6groups: model group was16mice; solvent group; ATRAgroup (10mg/kg); ATPR group (5mg/kg,10mg/kg,20mg/kg) was15mice in eachgroup.control group; model group; ATRA group (10mg/kg); ATPR group (5mg/kg,10mg/kg,20mg/kg), with15mice per group. Solvent group was injected withpolyoxyethylene castor oil; and dosing group started to inject corresponding doses ofdrugs the every other day for5continuous weeks intraperitoneally.3Efficacy evaluation: Observed animal time of death and recorded the survival daysof Statistics and calculated of life extension rate.4Other secondary efficacy evaluation:(1) Testing of transplanted tumor weight and volume.(2) Variation of cell morphology: the liver, spleen, kidney, lung and tumor of eachgroup were fixed with10%formaldehyde above48h, embedded in paraffin, cut sheet and stained hematoxylin-eosin (HE). Then observe the tissue morphology by amicroscope.(3) The paraffin sections of tumor tissue were dropped goat anti human COX-2/CEApolyclonal antibody and secondary antibody marked with biotin. Then observeexpression changes of COX-2and CEA under microscope.(4)Flow cytometry (FCM) detection of cells cycle of tumor: tumor tissues wereweighed and grinded, fixed overnight with70%ethanol in-20℃refrigerator, dyed30min with PI in dark. More than104cells were detected by flow cytometry.(5) Detection of serum alkaline phosphatase (ALP) and lactate dehydrogenase(LDH) activity: blood was taked from eyeball, standed and centrifugated. Thensupernatant was imbibed and both activity were tested ccording to alkalinephosphatase and lactate dehydrogenase kit manual.5Some study mechanism: using RT-PCR, Western Blot technique to detect theexpression changes of RARβ mRNA and protein induced by ATPR in SGC-7901cells.And we used a technique called RNA interference to target silence RAR β gene ofSGC-7901cell.6h later, we added ATPR(final concentration of10-5mol/L) in andobserved cell proliferation and the influence of the relevant enzyme activity. Weaimed to study the effect of RARβ in mediating ATPR on SGC-7901.Results：1. Tumor tissue suspension was injected into mouse armpit, after a week,subcutaneous mung bean white small protrusions can see, description model wassuccessfully established.2. Novel retinoid derivatives ATPR can prolong the survival of nude mice transplanted with tumor.3. Internal organs such as liver, spleen, kidney and lung of nude mice were comparedwith normal mice by microscope, but no significant pathological changes wasfound. The tumor cells in mice were round, big nucleus, deformity, arranged innests or cords under microscope; the tumor cells of model group and solventgroup have two or three nuclear in endoscopically, but a part of cells of mediumand high dose group were apoptosis.4. Positive material of cyclooxygenase-2(COX-2) immunohistochemistry locate inthe cytoplasm, brownish yellow or brown color and optical density was analysisby IPP software. COX-2expression of ATPR high dose group and ATRA positivemedicine group compared with model group and solvent group is decreasedobviously.5. CEA protein was expression in the cell membrane and cytoplasm, brown, and theoptical density was analysised by IPP software, CEA expression of the ATPRhigh-dose group and the ATRA positive group compared with the model groupand solvent group was reduced significantly.6. ATPR has the anti-proliferative effect of xenografts in nude mice; G0/G1phasecells of the drug group increased than other groups.7. ATPR has induce differentiation effect of xenografts in nude mice; thedifferentiation marker enzyme ALP and LDH assay showed the activity of ALPand LDH in treated groups were decline.8. MTT method and flow cytometry results showed that: RARβ gene of SGC-7901was targeted silence6hours later, treated with ATPR and ATRA for72h. The cell growth of siRNA-RARβ group, siRNA-RARβ+ATPR group and siRNA-RARβ+ATRA were significantly increased than ATPR group without silencing, but wereno significant difference compared with blank control group.9. Alkaline phosphatase and lactate dehydrogenase activity detection: both the twoenzyme activity of two tumor markers in RARβ transfection silent plus druggroup were significantly higher than direct dosing group.Conclusion:1. SGC-7901cell lines injected into nude mice could successfully establish gastriccarcinoma xenograft model.2. Novel retinoid derivatives ATPR can prolong the mice transplanted with gastriccancer cell survival period and decrease the activity of differentiation markerenzyme alkaline phosphatase, lactate dehydrogenase, and decrease the expressionof COX-2.3. Guessing that the mechanisms of inducing differentiation effect of ATPR iscombination with the RARβ receptor, and then specific binding to the regulatoryregion of target genes of retinoic acid response element (RARE), and therebyregulating the SGC-7901cells proliferation and differentiation.