Spectroscopic and Calorimetric Evaluation of Chemically Induced Protein Denaturation in HuH-7 Liver Cancer Cells and Impact on Cell Survival

Abstract

Solid tumors such as hepatocellular carcinoma are very often not amenable to chemotherapy and radiotherapy. Local ablation methods, including chemical ablation with absolute ethanol, are therefore an option for treatment but lack of information about the mechanism of devitalization leading to cell death is a hindrance to further adoption. Systemic toxicity also has limited the amount of ethanol that can be used in a single treatment session. Therefore we evaluated the mechanism of urea, a denaturant with little or no systemic toxicity, for potential use in chemical ablation. In this study we report on the use of three methods to analyze the effects in cell culture with a view towards eventual clinical application. Human hepatoma HuH-7 cells were analyzed at several time points after treatment using FTIR, DSC, and Raman microspectroscopy based on MTT and PI-exclusion viability assays. Time course fractional denaturation data plotted against viability show that a 50% viability drop occurs after only a 10-20% drop in overall protein denaturation. Other methods of cell death such as apoptosis may also be operative, but this result implies that protein denaturation is one of the major mechanisms of cell death. This is in line with what has been previously suggested for purely thermal methods, and opens the way to mechanism-based improvements in chemical ablation of solid tumors.

Correlated Parameter Fit of Arrhenius Model for Thermal Denaturation of Proteins and Cells

“…Then we validated our correlated parameter fit approach with literature data including denaturation of a pure protein (collagen peptides) [25] and a cell line (AT-1) [8] , and demonstrated the close agreement of our proposed protocol with the literature data. Finally, we demonstrated the utility of the correlated parameter fit on thermally induced denaturation of a pure protein (bovine serum albumin) and several cell lines (new data on HDF and LNCaP; historial data on AT-1 (ref# 8 and HuH-7 (ref# 5)) as measured by FTIR spectroscopy.…”

“…(heating rate: 2°C min −1 ). [5] Figure 6A shows the increase in the β sheet structures for these cell types as a function of temperature. First derivative plots (Figure 6B) are included to identify peaks and patterns in the denaturation profiles.…”

“…This process is important for a number of biotechnologies [2, 3] , disease conditions and treatments. [4, 5] Thermally induced protein denaturation, in particular, has found important applications in food processing, [3] burn injury, and thermal ablation of diseased or cancerous tissue, [6, 7] and has been proposed to correlate with cell injury. [4, 8] …”

Investigating the effects of Pentoxifylline on human breast cancer cells using Raman spectroscopy

“…As per the doctrine, it is the dose that determines the outcome of a biological process. In our study, we have therefore evaluated the e®ects of PTX at subtoxic doses i.e., doses exhibiting a maximal of 30% toxicity or till IC 30 . Higher doses have been excluded from the same in purview of this rationale.…”

“…19 It has the advantage of having minimum interference from water bands which makes it more appropriate for biomedical applications 20 and provides an easy, chemicalfree and nondestructive method for studying cells in vitro, cell drug interactions as well as for distinguishing neoplastic from normal tissues. [21][22][23][24][25][26][27][28][29][30][31][32] Each tumor is composed of several distinct cell populations and thus the potential and e±cacy of chemotherapy is largely governed by the presence of most chemosensitive or rather least resistant cell populations. Identi¯cation of a resistant/sensitive phenotype in cancer cells from the patients can enhance the e±cacy of therapy.…”