Abstract
Background:Autophagy is a catabolic process, utilized constitutionally by body cells to recycle nutrients and to remove unwanted/damaged intracellular constituents. It is enhanced during periods of stress, such as starvation and hypoxia, aiding in cell survival and it is linked to major cellular processes, such as apoptosis and antigen expression. The process has been extensively studied in vitro models or tumor tissue samples with rare application on human subjects. Methods:Plasma samples from 24 advanced solid tumor patients were collected at different time points before and after chemotherapy. Their exosomes were isolate and blotted for microtubule-associated protein-1 light chain-3 (LC-3B) protein as a marker for autophagy. All the subjects received a standard chemotherapy regimen of carboplatin- gemcitabine with chloroquine (CQ)/ hydroxychloroquine (HCQ) in chronic doses throughout their treatment period as an autophagy modulator. CQ/HCQ was given in 50 mg increments as guided by their tolerability to treatment. Results:A total of 267 plasma samples were obtained for the 24 patients and processed. Each sample corresponds to a single time point. The first group included 6 patients, all received 50 mg of CQ with chemotherapy. LC-3B I was detected in their isolated exosomes, while LC3-BII was not detected in their samples. The second cohort of patients included 3 subjects who re-ceived 100mg of HCQ. They demonstrated both LC3-BI and II on day 15 after chemotherapy in one patient, and on third cycle after 24 hours in the second patient. The third cohort included 3 subjects who received 150 mg of HCQ. All cases demonstrated LC3-BI and II on first cycle of treatment after less than 24 hours. The last cohort included 8 subjects, who received a fixed dose of 100 mg of HCQ with treatment. In this cohort, we were able to detect both LC3-B isoforms on advanced time points of second and third cycles. Conclusion:Detection of autophagy protein LC3-B in exosomes serves as a dynamic method to monitor autophagy. It can be utilized to study the effects of anti-neoplastic agents on autophagy and mechanisms of drug resistance, however, to standardize our results a larger specimen of patients should be included.
Highlights
Autophagy is a self-degradative process that occurs in eukaryotic cells to maintain protein he-mostasis and recycling of organelles
These time points were: 1h, 2h, 4h, 6h and 24h. Since these cells were not treated with any agents, we calculated the time points from the point when fresh exosome free media was added to the cell culture dishes at 60% confluency
Blotting the cells showed equal expression for LC3-BI and II for all cell lines at all time points, the bands were most clear at 24h (Figure 1.1b)
Summary
Autophagy is a self-degradative process that occurs in eukaryotic cells to maintain protein he-mostasis and recycling of organelles. Autophagy is a catabolic process, utilized constitutionally by body cells to recycle nutrients and to remove unwanted/damaged intracellular constituents It is enhanced during periods of stress, such as starvation and hypoxia, aiding in cell survival and it is linked to major cellular processes, such as apoptosis and antigen expression. Methods: Plasma samples from 24 advanced solid tumor patients were collected at different time points before and after chemotherapy Their exosomes were isolate and blotted for microtubule-associated protein-1 light chain-3 (LC-3B) protein as a marker for autophagy. The last cohort included 8 subjects, who received a fixed dose of 100 mg of HCQ with treatment In this cohort, we were able to detect both LC3-B isoforms on advanced time points of second and third cycles. It can be utilized to study the effects of anti-neoplastic agents on autophagy and mechanisms of drug resistance, to standardize our results a larger specimen of patients should be included
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