Antibody-based diagnosis and therapy of lung cancer targeting calnexin

Abstract

Lung cancer is the one of fatal diseases with high rate of death in the world, due to the absence of effective early diagnosis methods and therapeutics. Here, calnexin, which is a molecular chaperone, was validated as a potential target protein for lung cancer. Calnexin was the one of selected candidate proteins, which detected in only four types of lung cancer cell lines but not in normal lung tissue by 1D-LC-MS/MS analysis. To verify specific expression on the surfaces of lung cancer cell lines, western blotting analysis, flow cytometry and immunocytochemistry were performed. Higher expression of calnexin was observed in four types of lung cancer cell lines comparing to normal human lung epithelial cell line. Membrane expression of calnexin was also confirmed. Overexpression of calnexin in tumor region in lung compared to that normal area in lung was verified by immunohistochemistry. Then, we found that anti-CANX antibody has cytotoxicity against lung cancer cell lines through cytotoxicity assay. In addition, growth of tumor was effectively reduced in subcutaneous xenograft mice models by anti-CANX antibody treatment. Furthermore, we examined possibility of calnexin as a diagnostic marker by ELISA to detect exosomal calnexin in blood plasma from normal healthy control and lung cancer patients. There was significantly different expression level of exosomal calnexin in blood plasma from normal healthy control and lung cancer patients. In conclusion, we suggest that calnexin can be a potent candidate for diagnosis and therapeutic target for lung cancer. |폐암은 효과적인 초기 진단 방법과 치료법이 없어 전세계적으로 높은 사망률을 동반하는 치명적인 질병 중 하나이다. 폐암을 효과적으로 치료하고 진단하기 위한 표적 단백질로서의 molecular chaperone인 calnexin의 가능성에 대해 확인했다. Calnexin은 폐암 표적 단백질 후보 중 하나로, 1D-LC-MS/MS 분석을 통해 정상 폐 조직에서는 검출되지 않고 네 가지 종류의 폐암세포에서만 검출되는 것을 확인했다. CANX이 폐암 세포의 표면에 특이적으로 발현하는 것을 확인하기 위해 western blotting analysis, flow cytometry, immunocytochemistry를 진행했다. 정상 세포에 비해 폐암 세포에서 calnexin이 과발현 하는 것을 확인 할 수 있었고, calnexin이 폐암 세포의 표면에 발현하는 것을 확인했다. 또한, immunohistochemistry를 통해 정상 폐 조직에 비해 폐암 조직에서 calnexin의 높은 발현량을 확인했다. 다음으로, anti-CANX antibody의 lung cancer cell line에 대한 cytotoxicity에 대해 확인한 뒤, subcutaneous xenograft mice models에서 anti-CANX antibody에 의해 종양 성장이 효과적으로 억제 되는 것을 확인했다. 마지막으로 calnexin의 폐암 진단 마커로서의 가능성을 확인하기 위해, 정상인과 폐암 환자의 혈액 plasma에 존재하는 exosomal calnexin의 양을 ELISA를 이용해 측정했다. 정상인과 폐암 환자의 exosomal calnexin의 발현량 사이에 유의적인 차이가 나타남을 확인했다. 이러한 실험 결과를 통해 calnexin이 폐암의 진단 및 치료를 가능하게 할 잠재적 후보가 될 수 있을 것이라 생각한다.; Lung cancer is the one of fatal diseases with high rate of death in the world, due to the absence of effective early diagnosis methods and therapeutics. Here, calnexin, which is a molecular chaperone, was validated as a potential target protein for lung cancer. Calnexin was the one of selected candidate proteins, which detected in only four types of lung cancer cell lines but not in normal lung tissue by 1D-LC-MS/MS analysis. To verify specific expression on the surfaces of lung cancer cell lines, western blotting analysis, flow cytometry and immunocytochemistry were performed. Higher expression of calnexin was observed in four types of lung cancer cell lines comparing to normal human lung epithelial cell line. Membrane expression of calnexin was also confirmed. Overexpression of calnexin in tumor region in lung compared to that normal area in lung was verified by immunohistochemistry. Then, we found that anti-CANX antibody has cytotoxicity against lung cancer cell lines through cytotoxicity assay. In addition, growth of tumor was effectively reduced in subcutaneous xenograft mice models by anti-CANX antibody treatment. Furthermore, we examined possibility of calnexin as a diagnostic marker by ELISA to detect exosomal calnexin in blood plasma from normal healthy control and lung cancer patients. There was significantly different expression level of exosomal calnexin in blood plasma from normal healthy control and lung cancer patients. In conclusion, we suggest that calnexin can be a potent candidate for diagnosis and therapeutic target for lung cancer.