Oral Carbon Monoxide Enhances Autophagy Modulation in Prostate, Pancreatic, and Lung Cancers

Abstract

Modulation of autophagy, specifically its inhibition, stands to transform the capacity to effectively treat a broad range of cancers. However, the clinical efficacy of autophagy inhibitors has been inconsistent. To delineate clinical and epidemiological features associated with autophagy inhibition and a positive oncological clinical response, a retrospective analysis of patients is conducted treated with hydroxychloroquine, a known autophagy inhibitor. A direct correlation between smoking status and inhibition of autophagy with hydroxychloroquine is identified. Recognizing that smoking is associated with elevated circulating levels of carbon monoxide (CO), it is hypothesized that supplemental CO can amplify autophagy inhibition. A novel, gas-entrapping material containing CO in a pre-clinical model is applied and demonstrated that CO can dramatically increase the cytotoxicity of autophagy inhibitors and significantly inhibit the growth of tumors when used in combination. These data support the notion that safe, therapeutic levels of CO can markedly enhance the efficacy of autophagy inhibitors, opening a promising new frontier in the quest to improve cancer therapies.

Keywords: CO biofoams; autophagy modulation; combination therapies; smoking.

Figure 1

Active smokers treated with autophagy inhibitors had excellent outcomes during clinical trial. A) COHb % in whole blood measured using a blood gas analyzer of non‐smokers versus active smokers within 3 h of their last cigarette (n = 10 per arm, p < 0.0001). P values were determined by unpaired t test. B) Intention‐to‐treat analysis for overall response (OR) in active smokers versus all patients. C) Change in diameter of target lesions from baseline, with active smokers identified by each arrow. Subfigure (C) was modified with permission from JAMA Oncology.

Figure 2

Schematic illustrating how CO‐GeMs are administered in combination with autophagy inhibitors and how they reach their targets. CO induces autophagy and when combined with downstream autophagy inhibition results in enhanced cancer cell death.

Figure 3

CO‐GeMs achieve sustained, elevated COHb in both small and large animals. A) Pressurized vessel for creation of CO‐GeMs and macroscopic and microscopic images of CO‐GeMs. B) Concentration of CO delivered by GeMs and CO‐enriched lactated Ringer's solution (LR). Percentages of COHb measured in C) mice and D) pigs at the indicated time after oral CO‐GeM administration (n = 5 per group). The dotted line represents highest baseline COHb values. E) Organ‐specific concentrations of CO in mice at 15 min after CO‐GeM administration via oral gavage (5 g kg⁻¹). Data are means (n = 5 animals per arm, with each sample evaluated in triplicate). P values were determined by unpaired t test comparing tissue CO concentration between animals that received oral CO‐GeMs (CO) versus animals that received oral GeMs infused with room air (RA).

Figure 4

CO induces autophagy in cancer cells. A) Western blotting to assess levels of phosphorylated‐AMPKα, LC3, and cleaved caspase 3 in mouse prostate cancer cells (MyC‐CaP, left) and (PC3, right) exposed to CO or room air. Right: Quantification of Western blotting results for the same proteins in human prostate cancer cells (PC3). Western blots are representative images from 3 independent experiments. B) Autophagic flux in cells transduced with an adenovirus coding for GFP‐RFP‐LC3 CO versus room air. Red arrows indicate autophagosomes. C) Expression of the lysosomal marker LAMP2 in MyC‐CaP cells exposed to CO or room air. D) Proposed model for how CO exposure induces autophagy and the impact of combined autophagy inhibition. We show that exogenous CO increases phosphorylation of AMPK and mitochondrial ROS as previously shown (23). Both mechanisms lead to an increase in autophagy. E) Quantification of autophagosomes from subfigure B. P values were determined by one‐way ANOVA. Results represent mean ± SD of 3 independent experiments.

Figure 5

Co‐administration of CO and autophagy inhibitors are synergistic in cancer cell death. A) Cytotoxicity, as determined by crystal violet staining, of MyC‐CaP cells exposed to increasing doses of the autophagy inhibitors chloroquine (CQ) and Lys05 ± CO (250 ppm). Control groups were exposed only to the inhibitors in standard incubator conditions (5%CO₂) without CO. B) Quantification of the viability (IC₅₀ values) of PC3 and MyC‐CaP cells as a function of CQ and Lys05 dose. C) Quantification of cell viability for PC3 and MyC‐CaP cells subjected to shATG‐mediated knockdown of ATG5. Controls were non‐transduced cells exposed to 250 ppm CO. D) Western blotting showing levels of ATG5 protein in the cells shown in C. P values were determined by one‐way ANOVA. Results represent mean ± SD of 3 independent experiments. Western blots are representative of 3 independent experiments.

Figure 6

Anti‐tumor effects of CO‐GeMs and hydroxychloroquine (HCQ) are enhanced when they are combined. A) Tumor volume in human prostate cancer xenografts treated with CO‐GeMs and HCQ, alone and in combination. Result represent mean± SD of 7 mice/group B) Histological staining of representative tumors in (A) for H&E and cleaved caspase 3. C) Quantification of tumor volume in murine syngeneic model of prostate cancer following treatment with CO‐GeMs and HCQ, alone and in combination. D) Quantification of cleaved caspase 3‐positive areas analyzed in randomly selected 600 µm x 600 µm sections of tissue at full thickness t (×8 magnification). Data represent means (n = 7 mice/group, three replicates per mouse). P values were determined by one‐way ANOVA. * – p < 0.05; ** – p < 0.01.