Analyzation Of Highly Active ALDH Isoforms In Gastric Cancer Cell Lines And Its Expression Profile In Gastric Cancer Tissue

It is well known that tumors are consist of heterogenous groups, As a small subpopulation of tumor cells, cancer stem cells (CSCs) exhibit a self-renewal capacity, resistant to conventional chemotherapy and are responsible for therapy failure, cancer relapse and metastasis. Increasing evidences suggest that enhanced Aldehyde dehydrogenases(ALDHs) activity might be a hallmark of cancer stem cells, for the members of this family play an important role in retinol acid signaling,which could lead to the differentiation,apoptosis and cell cycle arrest in normal cells and gain stemness characteristics in tumor cells. The ALDH enzymes are family comprised of19isoforms and recent studies have demonstrated that its role as a cancer stem cell marker come down to specific isoform. Although ALDH+gastric cancer cells have been demonstrated to be possessed CSC properties, the specific isoform responsible for ALDH activity in gastric cancer has not been determined.We therefore analyze the highly active ALDH isoforms in gastric cancer cell lines and the expression profile in gastric cancer tissue.Main methods as follows:1. The identification of stemness characteristics of ALDH+gastric cancer cells.1) The ALDH+gastric cancer cells would be achieved from the cell line MKN-45 and SGC-7901and cultured in serum free condition medium.2) The stemness genes such as Bmi-1,Sox-2and Oct-4were examined by qRT-PCR and Western blot in the ALDH+cells and ALDH-cells.3) The ALDH+gastric cancer cells were cultured in serum free medium and passasaged for at least3consecutive generations.4) The ALDH+gastric cancer cells were then cultured in medium supplemented with10%FBS to induce the differentiation.5) The same amount of ALDH+and ALDH-cells were transplanted to NOD/SCID mice to compare the tumorigenic ability.2. Identification of the highly active ALDH isoforms in ALDH+subgroup. The expression of19isoforms in ALDH family was analyzed by real time PCR. ALDH3A1was knockdowned by si-RNA to examine its role in maintaining the ALDH+subgroup.3. Exploration of the expression profile of ALDH3A1in gastric cancer tissue. Immunohistochemistry (IHC) was used to detect the expression of ALDH3A1in25specimens of gastric cancerMain results as follows:1.The ALDH+gastric cancer cells possesses stemness characteristics.1) The ALDH+gastric cancer cells could be achieved from the cell line MKN-45and SGC-7901; The ALDH+cells could grow as tumor spheres in serum free condition medium.2) Compared with ALDH" gastric cancer cells, the ALDH+cells highly expressed the stemness genes such as Bmi-1,Sox-2and Oct-4.3) The ALDH+gastric cancer cells could form tumor spheres for at least3consecutive passages.4) Cultured the ALDH+gastric cancer cells in medium supplemented with10%FBS, the expression of stemness marker CD44recruit to a level similar to ALDH+ cells.5) Xenografts assay showed that the ALDH+cells were highly tumorigenic.2.ALDH3A1was found highly expressed in ALDH+subgroup. The expression of19isoforms in ALDH family was analyzed by real timePCR.ALDH3A1,ALDH2,ALDH16A1and ALDH18A1were found highly expressed among which ALDH3A1increased the most.ALDH3A1was proved to be the key isoform in maintaining the aldefluor activity.3.The expression of ALDH3A1correlate with the grade of differentiation. Immunohistochemistry (IHC) was used to detect the expression of ALDH3A1in92specimens of gastric cancer, the results showed that the expression of ALDH3A1was correlated with the level of the differentiation grade, indicating that ALDH3A1might be an indicator of poor prognosis in gastric cancer.The conclusion as follows:1. ALDH+gastric cancer cells possess the stemness properties to some extent;2. ALDH3A1was highly expressed in ALDH+subgroup and could attribute to the aldefluor activity in gastric cancer cell lines;3. The expression of ALDH3A1in gastric cancer was correlated with the grade of differentiation and could potentially be a prognostic indicator.