The changing 50% inhibitory concentration (IC50) of cisplatin: a pilot study on the artifacts of the MTT assay and the precise measurement of density-dependent chemoresistance in ovarian cancer.
Inconsistencies in the half-maximal (50%) inhibitory concentration (IC50) data for anticancer chemotherapeutic agents have yielded irreproducible experimental results and thus reciprocally contradictory theories in modern cancer research. The MTT assay is currently the most extensively used method for IC50 measurements. Here, we dissected the critical reasons behind MTT-dependent IC50 inconsistencies. We showed that IC50 errors caused by the technical deficiencies of the MTT assay are large and not adjustable (range: 300-11,000%). To overcome severe MTT artifacts, we developed an unbiased direct IC50 measurement method, the limiting dilution assay. This detection technique led us to the discovery of the inherent density-dependent chemoresistance variation of cancer cells, which is manifold and unpredictable in its forms. The subsequent intracellular signaling pathway analysis indicated that pAkt and p62 expression levels correlated with alterations in the IC50 values for cisplatin in ovarian cancer, providing an explainable mechanism for this property. An in situ pAkt-and-p62-based immunohistochemical (IHCpAkt+p62) scoring system was thereby established. Both the limiting dilution assay and the IHCpAkt+p62 scoring system accurately predicted the primary chemoresistance against cisplatin in ovarian cancer patients. Furthermore, two distinct chemoresistant recurrence patterns were uncovered using these novel detection tools, which were linked to two different forms of density-chemoresistance relationships (positively vs. negatively correlated), respectively. An interpretation was given based on the cancer evolution theory. We concluded that the density-related IC50 uncertainty is a natural property of the cancer cells and that the precise measurement of the density-dependent IC50 spectrum can benefit both basic and clinical cancer research fields.
Department of Obstetrics and Gynecology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.|Shanghai Key Laboratory of Gynecologic Oncology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.|Tumor Microenvironment and Metastasis Program, The Wistar Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Yifeng
Y
He
Department of Obstetrics and Gynecology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.|Shanghai Key Laboratory of Gynecologic Oncology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.|State Key Laboratory of Oncogene and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.
Qiujing
Q
Zhu
Department of Gynecology, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China.
Mo
M
Chen
Tumor Microenvironment and Metastasis Program, The Wistar Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Qihong
Q
Huang
Department of Obstetrics and Gynecology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.|Shanghai Key Laboratory of Gynecologic Oncology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.|State Key Laboratory of Oncogene and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.
Wenjing
W
Wang
Department of Obstetrics and Gynecology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.|Shanghai Key Laboratory of Gynecologic Oncology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.|State Key Laboratory of Oncogene and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.
Qing
Q
Li
Children's Research Institute, Children's National Medical Center, Washington DC 20010, USA.
Yuting
Y
Huang
Department of Obstetrics and Gynecology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.|Shanghai Key Laboratory of Gynecologic Oncology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.|State Key Laboratory of Oncogene and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.
Wen
W
Di
D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D016477:Artifacts; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology* / Q000627:therapeutic use; D003124:Colorimetry / Q000379:methods; D019008:Drug Resistance, Neoplasm; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D020128:Inhibitory Concentration 50; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000473:pathology; D010053:Ovary / Q000473:pathology; D010766:Phosphorylation; D010865:Pilot Projects; D061251:Primary Cell Culture; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
D000970:Antineoplastic Agents; C118585:P62 protein, human; D016601:RNA-Binding Proteins; D051057:Proto-Oncogene Proteins c-akt; D002945:Cisplatin
50% inhibitory concentration; cisplatin; density; inconsistency; ovarian cancer
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