All-trans Retinoic Acid (ATRA)'s Impact On Colorectal Cancer Cells And Their Connexin 32,Connexin 43

Background and purpose:Colorectal cancer is the third most commonly diagnosed cancer in males and the second in females, with over 1.2 million new cancer cases and 608,700 deaths estimated to have occurred in 2008.Treatment modality of colorectal cancer is surgery followed by multidisciplinary systemic therapy and appropriate combination chemotherapy is the essential component, which has been validated to considerably ameliorate colorectal cancer patients'survival. However, cure rates remain low and the 5 year survival of metastatic colorectal cancer is only 11%. Therefore, it is an urgent need and of great clinical value to exploit a new regimen.All-trans retinoic acid(ATRA)is the active metabolite of vitamin A that belongs to retinoid family and in combination with chemotherapy, has made a spectacular achievement in treatment of acute promyelocytic leukemia(APL). In vitro, ATRA could also function in such solid cancers as esophageal cancer, cervical cancer, gastric cancer, CNS tumors. It targets cancer cell survival related signal pathways, resulting in proliferation suppression, cancer stem cells differentiation, protein expression alteration and eventually the transformation of cancer cells' biological behaviours.Gap junction(GJ) is one of the intercellular junctions (Desmosome, Adherens junction,Tight junction and Gap junction), which plays a key role in such physiological processes as cell proliferation and differentiation, homeostasis, metabolism and growth control, with gap jnctional intercellular communication (GIJC) the major mechanism. Connexins are the basic units of GJ and lay the molecular foundations of GJ. There is a tight connection between colorectal cancer and connexins, of which,Cx43 and Cx32 are extensively being researched at present. Membranous distribution of Cx43 and Cx32 was diminished in colorectal cancer cells, with incremental cytoplasmic accumulation, which may lead to impaired GIJC and alteration of intercellular communication, eventually favoring cancer cell invasion and migration.In view of the state-of-the-art of researches on ATRA in solid cancers and the important role of connexins in colorectal cancer, here we tentatively investigate ATRA's impact on proliferation and migration of colorectal cancer cells SW480 and CaCO2, and expression alteration of membranous Cx32 and Cx43 to try to provide a clue for further researches about ATRA's effects on colorectal cancer'biological behaviours and their potential mechanisms..Methods:The logarithmic growth period colorectal cancer cell lines Caco-2 and SW480 were divided into ATRA treatment groups and control groups. With the 1×10-8mol·L-1, 1×10-7 mol·L-1, 1×10-6 mol·L-1,1×10-5 mol·L-1 and 1×10-4 mol·L-1 ATRA treated on Caco-2 and SW480 cells,MTT assay was applied to gauge colorectal cancer cells CaCO2 and SW480 proliferation inhibition rate; With the 1×10-5 mol·L-1 and 1×10-4 mol·L-1 ATRA treated on SW480 cells, Wound healing assay was employed to determine the cell migration ability and flow cytometry was used to analyze membranous distribution of connexin 43 and 32 in SW480 cells after treated with ATRA.Results:There was no obvious impact on Caco-2 and SW480 cells at the concentration 1×10-8 mol·L-1～1×10-5 mol·L-1,whereas colorectal cancer cells' proliferation was significantly inhibited at the concentration 1×10-4 mol·L-1,with growth inhibition rate was 54.90±6.98%,84.19±4.14% and 87.58±8.03% for Caco-2 cells and 56.72±6.65%.76.71±3.86% and 83.71±10.30% for SW480 cells respectively,24,48 and 72h after treatment. And SW480 cells'migration capacity was dramatically diminished by 1×10-5 mol·L-1 ATRA, with the mean migration rate 8.97±4.36% and 21.85±5.75%,24 and 48h after treatment, lower than the control group(41.90±5.24% and 67.25±6.60%, respectively). In terms of membranous distribution of connexin 32 and 43,48h after treatment,Cx32 membrane positive rate shifted from 59.89±0.78399% to 72.31±4.02%(p<0.05) and 78.88±3.00%(p<0.05) respectively at the ATRA concentration 1×10-5 mol·L-1 and 1×10-4 mol·L-1, with Cx43 form 40.55±9.70% to 64.03±1.59%(p<0.05) and 66.33±2.77%(p<0.05)Conclusion:ATRA could markedly inhibit the proliferaion of colorectal cancer cells CaCO2 and SW480 in high concentration (1×10-4 mol·L-1) whereas 1×10-5 mol·L-1 ATRA diminished migration capability of SW480 cells in vitro and upregulated membranous expression of Cx43 and Cx 32,which may be one of the potentil mechanisms contributing to impaired migration capability of SW480 cells resulting from ATRA.