Zhou, Chenghui 
ORCID: 0000-0003-1170-1761
(2021).
Aldo-keto reductase 1C3 mediates chemotherapy resistance in esophageal adenocarcinoma via ROS detoxification.
Diploma thesis, Universität zu Köln.
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Abstract
Background:
The incidence and mortality of esophageal adenocarcinoma (EAC) are very high, and the prognosis is poor in Europe. During the past years, Aldo-keto reductase 1C 3 (AKR1C3) as an oxidoreductase has attracted much attention in the field of cancer research. Especially in the treatment of tumors, AKR1C3 seems to show its protective effect on tumor therapy in many cancers. However, whether its role in the treatment of EAC is still unknown.
Methods:
Public datasets were downloaded and applied. Western blot (WB) was used to check the protein level. Immunofluorescence was also used to measure the expression and localization of AKR1C3. Cell lines in SKGT-4 (SKGT-4AKR1C3-KD /SKGT-4AKR1C3-shNT) and OACP4C (OACP4CAKR1C3-KD /OACP4CAKR1C3-shNT), and cell lines in OE33 (OE33AKR1C3-OE /OE33AKR1C3-VEC) and FLO-1 (FLO-1AKR1C3-OE /FLO-1AKR1C3-VEC) were established. Basic cell experiments such as cell proliferation/colony formation/wound healing assay were carried out in this study on those genetically modified EAC cell lines. MTT assay was used to quantify the cells’ viability after anti-cancer drugs treatment. Then cisplatin-induced cell apoptosis was checked by FACS using Annexin V/DAPI. Reactive oxygen species (ROS) of EAC cells were checked by FACS using H2DCFDA. Chromatin-immunoprecipitation (ChIP) was carried out to validate the transcriptional regulation of AKR1C3. SKGT-4NRF2-KD and SKGT-4NRF2-shNT cell lines were established. Then, PCR and WB were used to check its level in SKGT-4NRF2-KD and SKGT-4shNT cell lines. GSH quantification of genetically modified EAC cells was also checked. Moreover, mitochondrial stress test was performed in evaluating oxygen consumption rate (OCR) of EAC cells.
Results:
AKR1C3 mRNA level in EAC tissues is higher than in the normal squamous esophageal epithelium in the data of GSE26886 and GSE92396 from public database. However, only about half of the 12 patients’ AKR1C3 protein level had elevated in tumor tissue than in matched normal tissue. Although public databases revealed the trend that higher AKR1C3 means poorer survival probabilities, no difference in survival can be observed between the AKR1C3high and AKR1C3low group. Then, we detected its protein level in OE19, OE33, OACP4C, FLO-1, JHesoAD1 and SKGT-4 cell lines. It high-expressed in OE19, OACP4C and SKGT-4 cell lines, and it low-expressed in OE33, JHesoAD1 and FLO-1 cell lines. Immunofluorescence showed the same results with WB. Furthermore, immunofluorescence results also found that AKR1C3 was mainly expressed in the cytoplasm.
OE33AKR1C3-OE and FLO-1AKR1C3-OE cell lines were more capable of proliferating/ colony-forming/migrating than OE33AKR1C3-VEC and FLO-1AKR1C3-VEC cell lines. Opposite results were observed in SKGT-4AKR1C3-KD and OACP4CAKR1C3-KD cell lines. Moreover, OE33AKR1C3-OE and FLO-1AKR1C3-OE cells had less apoptosis induced by anti-cancer drugs, while SKGT-4AKR1C3-KD and OACP4CAKR1C3-KD cells showed the opposite results. Furthermore, we found that higher ROS in SKGT-4AKR1C3-KD and OACP4CAKR1C3-KD cells and lower ROS in OE33AKR1C3-OE and FLO-1AKR1C3-OE cells compared with their control cell lines. The nuclear factor erythroid 2-related factor 2 (NRF2) controls redox homeostasis, and it binds with AKR1C3’s promoter and mediates its expression. And their expression was decreased in SKGT-4NRF2-KD cells than in the SKGT-4NRF2-shNT cells. Moreover, the rescue experiment showed that NAC and BSO could make AKR1C3’ effect disappear whether in SKGT-4/OACP4C AKR1C3-KD/shNT cells or OE33/ FLO-1AKR1C3-OE/VEC cells. Furthermore, the phosphorylation of AKT was decreased in SKGT-4AKR1C3-KD and OACP4C AKR1C3-KD cell lines than in SKGT-4AKR1C3-shNT and OACP4CAKR1C3-shNT cell lines. Conversely, it was increased in OE33 AKR1C3-OE and FLO-1 AKR1C3-OE cell lines than OE33 AKR1C3-VEC and FLO-1 AKR1C3-VEC cells lines. AKT inhibitor could inhibit the phosphorylation of AKT but had no effect on AKR1C3 expression. And inhibitor of AKT could diminish the protective effect of the decrease for apoptotic cells caused by the elevated of AKR1C3 in OE33AKR1C3-OE and FLO-1AKR1C3-OE cell lines. Moreover, GSH levels was decreased, and the inhibitor of AKT had the same effect with AKR1C3-KD for its regulation. We found that the OCR is increased in OE33 AKR1C3-OE than in OE33AKR1C3-VEC, while SKGT-4AKR1C3-sh1 and SKGT-4AKR1C3-sh2 showed the opposite results than SKGT-4AKR1C3-shNT. Lipid metabolism substances such as fatty acids, steroids were enriched in AKR1C3high group from public databases
Conclusions:
AKR1C3 regulates chemotherapy of EAC and may contribute to chemotherapy resistance of tumor cells. The possible mechanism is that the elevated AKR1C3 enhanced its own antioxidant capacity by reducing the ROS levels through AKT/GSH signaling. Tumor cells were exposure in chemotherapy drugs, an abundance of ROS will be produced in tumor cells, while the cells with higher AKR1C3 expression in the cells will show a stronger antioxidant capacity against ROS and thus be more likely to survive. So, this study provides rationale for the design of remedy that targeting AKR1C3 might enhance the anti-tumor effect and prolong survival time of EAC patients.
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