The Effects Of 4-amino-2-trifluoromethyl-phenyl Retinate On Human Gastric Cancer Cell Line BGC-823 And Possible Mechanisms

Background Globally, although the incidence and mortality of gastric cancer has been observed the overall downward trend in the past seven decades, the gastric cancer is still the fourth most common cancer, and ranked the second in the list of death caused by cancer. Meanwhile, gastric cancer is also the one of the most common cancer in Asia, and showed upward trend in China. After surgery to the patients with early gastric cancer, some patients still needed to receive the chemotherapy to prevent local recurrence or distant metastasis. As for advanced or diffuse-type gastric cancer, patients had not many options except chemotherapy. However, there is not good program of chemotherapy in clinic for these patients with gastric cancer. So new non-toxic anti-cancer drugs with highly effectiveness and acceptable standard chemotherapy program is urgent in the present clinical operation.All-trans retinoic acid(ATRA) is the first line drug to treat acute promyelocytic leukemia. ATRA has not been widely applied clinically due to some adverse effects, such as retinoic acid syndrome and retinoic acid resistance, which also drove researchers to obtain more potent and less toxic derivatives by changing the ATRA structure. In my lab, we had synthesized a series of derivatives of ATRA, and found that 4-amino-2-trifluoromethyl-phenyl retinate(ATPR) can perform better anti-cancereffects in many kinds of tumours. But we do not clear that whether ATPR can inhibit the human poorly differentiated gastric cancer cell line BGC-823.Previous studies depicted that ATRA could perform its anti-cancer effects by binding its receptors, such as retinoic acid receptors and retinoic acid X receptor, followed by regulating the transcription of target genes, which could induce differentiation or inhibit proliferation of the cancer cells. But we don’t know whether ATPR also has the similar mechanism in its anti-cancer effects.The changes in cell adhesion and motility between tumor cells play an important effect in the process of tumor invasion and metastasis. The example is that tight junctions are one of the types of intercellular junctions; if the abnormality occurred in these junction, such as the structure or function of Occludin and ZO-1, which are the main protein in the junctions, changed to abnormal because of some environmental stress or inducer, which could result in the decreasing of the cell adhesion and facilitate cancer cells invasion and metastasis. As for the migration of the cancer cells, the phosphorylation of MLC II can activate myosin, trigger the glide between actomyosin and cause the migration of cells. MLCK, ROCK and MLCP three enzymes can adjust the phosphorylation of MLCⅡ to control cell migration. But we don’t know whether ATPR can affect the adhesion and motility of tumor cells to inhibit the cell migration.In recent years, the relationship of obesity, diabetes mellitus and cancer has drawn more and more concerns. Obese persons have more chances to suffer some certain cancers, and worse prognosis than the persons with normal body weight. The patients with obesity or diabetes often have the signs of insulin resistance, which make the body secret more compensatory insulin to form hyperinsulinemia. So at the last part of the present study, we explored whether the insulin would improve the migration of gastriccancer cells BGC-823 and ATPR would against this improvement.It has not been fully understood of the effect of ATPR to the biological behavior of gastric cancer cells, so we conducted the experiments to seek the possible roles of ATPR on poorly differentiated gastric cancer cell line BGC-823 and its mechanism. This study also could supply more theoretical foundation for the clinical application of ATPR in the future.Objectives To explore the possible mechanism, we evaluated the effects of ATPR to the proliferation, differentiation and migration of human gastric cancer cells BGC-823 compared with ATRA.Methods In BGC-823 human poorly differentiated gastric cancer cells, we firstly detected the cell proliferation after administration of ATPR using inverted phase contrast microscope, and then cell viability and IC50 were measured by MTT assay. Plate colony formation assay was applied to depict the ability of these cells to form colony in vitro. As for the differentiation after administration of ATPR, we observed the morphology changes under an inverted phase contrast microscope, followed by flow cytometry to detect cell cycle distribution, meanwhile analyzed ALP and LDH activity.Wound healing assay was applied to test the migration of BGC-823 cells with or without administration ATPR.In the second part of the present study, we detected the expression of the protein RARs, RXRs, MLCK, p MYPT1/MYPT1, p MLC/MLC, Occludin, ZO-1 using Western blots. The relative m RNA levels of MLCK, ROCK1, and ROCK2 were detected by real-timequantitative RT-PCR. As for the intracellular location of Occludin and ZO-1, we applied immunofluorescence to perform.Wound healing assay was also used to show the effects of specific inhibitors ML-7 and H-1152 to BGC-823 cells. Western blots were applied to detect the expression levels of the protein MLCK, p MYPT1 and MYPT1.At the same time, we analyzed the effects of insulin on BGC-823 cells using the similar methods to detect the proliferation and migration. In wound healing assay, we joint insulin and ATPR to observe ATPR’s influence on insulin effect. Western blots were applied to detect the expression levels of the protein MLCK, p MYPT1 and MYPT1.Results We observed that ATPR had more potent inhibition to BGC-823 cells at the same concentration compared with ATRA. This kind of inhibition was time-dependent and concentration-dependent. In the plate colony formation tests, we found the ability of colony formation was decreasing, which means the proliferation and adhesion of BGC-823 were impaired by 25 μmol/l ATPR treated for 48 h.After 25 μmol/l ATPR treated for 48 h, morphological differentiation of the cells was detected, and cell cycle arrest at the G0-G1 phase and S phase shortened, as well as the decreased activity of enzymes ALP and LDH. These results showed that ATPR had more effective to induce differentiation of BGC-823 cells than ATRA.Wound healing assays showed that 10 μmol/l ATPR could inhibit the migration of BGC-823 cells more effective than ATRA after treated for 48 h.In the following studies, we observed the expression of RARα, RARβ, RXRβ, MLCK,p-MYPT1 and p-MLC protein was decreased after administration of ATPR to BGC-823 cells. Meanwhile, the relative m RNA levels of MLCK, ROCK1 and ROCK2 were significantly decreased. While after administrated the inhibitor of MLCK, ML-7, or the inhibitor of ROCK, H-1152, the expression of MLCK and p-MYPT1 was downregulated and the migration of BGC-823 cells also was impaired. So we could come to a conclusion that the ATPR could inhibit the migration of BGC-823 cells by the inhibition of the activity of MLCK and ROCK.Besides, we observed the expression of tight junction-associated protein Occludin and ZO-1 was barely changed, but the location of these two kinds of enzymes was shifted to the cell surface.Regarding the effect of insulin, we found there would no effect on the proliferation of BGC-823 cells at the concentration of 0.5 ng/ml. If the concentration increased to 10 ng/ml or more, cell proliferation was improved, obvious in the meantime, pseudopodia and enhanced cell motility were observed. Interesting, we detected insulin could improve the migration of BGC-823 cells but ATPR could reverse this improvement.Conclusion ATPR, the new all-trans retinoic acid derivatives, could inhibit the proliferation, induce the differentiation and reduce the migration of human poorly differentiated gastric cancer BGC-823 cells.Under the same concentration and the same work time, ATPR was more efficient in the inhibition of the proliferation, induction of differentiation and the decrement of migration compared with ATRA.Meanwhile, ATPR could reduce the cell motility and improve the cell adhesion to inhibit the migration of the BGC-823 cells.And ATPR could regulate the biological behaviors using the downregulation of theretinoic acid receptor-MLCK/ROCK-p MLC signaling pathway.Also ATPR could inhibit MLCK enzyme activity to reverse the effect of insulin, which could improve the proliferation and migration of BGC-823 cells.