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

Abstract

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.

Glucosamine decreases the stemness of human ALDH+ breast cancer stem cells by inactivating STAT3

“…The results from the western blot analysis confirmed that glucosamine significantly inhibited STAT3 phosphorylation of CSCs and MCF7 cells to a similar degree. The less significant inhibitory effect of 4 mM glucosamine in ALDH + breast cancer cells may be due to the increased abundance of CSCs in ALDH + breast cancer cells, compared with MCF7 cells, which exerted a density-dependent effect on chemoresistance, as indicated by He et al (35). CSCs may also attempt to maintain their stemness by activating other pathways that contribute to STAT3 activation, including overactivation of the G-protein-coupled receptor signaling pathway or overexpression of gp130 (36).…”

CA125 over‐release behavior following a 75‐g oral glucose test as a predictive biomarker of multidrug resistance in patients with ovarian cancer

“…The concentrations of glucose (11.2 mM) and insulin (100 nM) administered to cultured ovarian cancer cells (HO8910, ES-2 and OVCAR-3) were comparable to the peak levels of blood glucose and insulin emerging between 0.5 and 1.0 h following 75-g oral glucose uptake. 26,27 We adopted a limiting dilution-modified clonogenic assay 5,25 to assess the 50% inhibitory concentrations (IC 50 s). A notable right-shift in the dose-response curves for both cisplatin and taxol was observed in cells pretreated with glucose and insulin for 1 h (Fig.…”

“…These findings can help to improve the current clinical limitations in ovarian cancer treatment, such as the delayed prediction of chemoresistance, a semiblind choice of chemotherapeutic agents, and thereafter, the loss of optimal treatment opportunities, which are frequently encountered by simply using tumor volume, IHC biomarkers or PDX models for evaluating or preventing chemoresistance. [4][5][6][7] CA125 has been a classical biomarker of ovarian cancer for over 30 years. However, few studies have focused on the significance of physiological/pathological fluctuations in this biomarker.…”

“…Clonogenic assays 25 were used to depict dose-response curves for cisplatin (Sigma-Aldrich) and taxol (Sigma-Aldrich) with a modification as described by He et al 5…”

“…If the cancer is prone to developing chemoresistance, an optimal debulking (residual site ≤1 cm) procedure is required, while a palliative resection could lead to the short-term occurrence of refractory cancer or rapid chemoresistant recurrence. However, apart from some specimen-based techniques, e.g., immunohistochemistry (IHC) of chemoresistance-related proteins, sequencing chemoresistance-related genes and patient-derived xenograft (PDX) models, [4][5][6][7] there are few convenient, noninvasive tools to presurgically evaluate the chemoresistance potential of ovarian cancer.…”