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. 2024 Apr 17;16(8):1525.
doi: 10.3390/cancers16081525.

Dichloroacetate and Quercetin Prevent Cell Proliferation, Induce Cell Death and Slow Tumor Growth in a Mouse Model of HPV-Positive Head and Neck Cancer

Yongxian Zhuang  1 , Joseph D Coppock  1 , Allison B Haugrud  1 , John H Lee  1 , Shanta M Messerli  1 , W Keith Miskimins  1
Affiliations

Dichloroacetate and Quercetin Prevent Cell Proliferation, Induce Cell Death and Slow Tumor Growth in a Mouse Model of HPV-Positive Head and Neck Cancer

Yongxian Zhuang et al. Cancers (Basel). .

Abstract

Elevated glucose uptake and production of lactate are common features of cancer cells. Among many tumor-promoting effects, lactate inhibits immune responses and is positively correlated with radioresistance. Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase that decreases lactate production. Quercetin is a flavonoid compound found in fruits and vegetables that inhibits glucose uptake and lactate export. We investigated the potential role and mechanisms of DCA, quercetin, and their combination, in the treatment of HPV-positive head and neck squamous cell carcinoma, an antigenic cancer subtype in need of efficacious adjuvant therapies. C57Bl/6-derived mouse oropharyngeal epithelial cells, a previously developed mouse model that was retrovirally transduced with HPV type-16 E6/E7 and activated Ras, were used to assess these compounds. Both DCA and quercetin inhibited colony formation and reduced cell viability, which were associated with mTOR inhibition and increased apoptosis through enhanced ROS production. DCA and quercetin reduced tumor growth and enhanced survival in immune-competent mice, correlating with decreased proliferation as well as decreased acidification of the tumor microenvironment and reduction of Foxp (+) Treg lymphocytes. Collectively, these data support the possible clinical application of DCA and quercetin as adjuvant therapies for head and neck cancer patients.

Keywords: Dichloroacetate (DCA); head and neck oral cancer; human papilloma virus; lactate; quercetin.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
DCA and quercetin inhibit mTOR signaling and have synergistic effects on inhibition of cell proliferation. For colony formation assays, MEER cells were plated on 100 mm dishes. The next day, cells were treated with the indicated treatments for one week, followed by staining with crystal violet. Representative images are shown in (A), and the corresponding average colony number as well as the p value compared with the control is plotted in (B). For cell cytotoxicity assays, cells were plated on 96-well plates and treated with either DCA, quercetin, or their combination. After two days, cells were stained with sytox green nucleic acid stain, and percentages of dead cells and possible synergy were analyzed using CalcuSyn software (Version 2.1) (C). (C) CI values at ED 75 and ED 90 demonstrated synergistic effects of the combination of DCA and quercetin. Western blotting was performed for levels of downstream targets of mTOR: p-S6, total S6, and 4EBP1, with actin serving as an internal loading control, and blots are shown in (D). Entire original blots are shown in Supplementary Materials Figure S1.
Figure 2
Figure 2
MEER cells were treated with DCA (5 mM), quercetin (25 µM), or their combination for one day. (A) Light microscopy images were taken at 100×. (B) Percentages of dead cells were determined using trypan blue cell exclusion. (C,D) Western blotting was performed for detection of PARP, cleaved PARP, and cleaved caspase 3. Actin was blotted as a loading control. Ratios of cleaved to uncleaved protein or cleaved protein to actin are plotted below their respective blots. The complete uncropped blots are shown in Supplementary Materials Figure S2.
Figure 3
Figure 3
MEER cells were treated with DCA (5 mM), quercetin (25 μM), or their combination for one day. Cells were then stained with Mito SOX (10 μM) for ten minutes, trypsinized, and resuspended for flow cytometry. Representative histograms of Mito SOX staining are shown in (A) and mean fluorescent intensity of the replicates is shown in (B). Western blotting for p-H2AX showed correlative increases in DNA damage with DCA, quercetin, and more so, their combination, as shown in (C). When N-acetyl cysteine (10 mM), a ROS scavenger, was added to DCA/quercetin-cotreated cells, the increases in ROS observed by the drug combination were partially rescued, as shown in (D,E). Western blotting showed correlative decreases in both DNA damage and induction of apoptosis via p-H2AX and cleaved PARP, respectively, in NAC-treated cells compared with the combination treatment of DCA/quercetin alone, as shown in (F). The entire uncropped blots are shown in Supplementary Materials Figure S3.
Figure 4
Figure 4
Culture media pH was measured after 4 h of the indicated treatments. (A) Change in pH from time zero was measured using a pH meter, and (B) lactate concentration in the media was measured using a commercially available lactate assay kit as described in the Materials and Methods. DCA, quercetin, and more so, their combination, increased media pH and decreased lactate concentration at 4 h. Calculated p values are listed above error bars in graphs, with c highlighting the control treatment.
Figure 5
Figure 5
MEER cells were injected into the subcutaneous right flank of C57Bl/6 mice and treated with indicated treatments for three weeks concurrent with once weekly chemoradiation also for three weeks. Tumor volumes were monitored weekly by measuring the tumor dimensions. Growth curves are shown in (A), indicating that the combination of DCA/quercetin has the greatest effect on tumor growth. (B) Survival plot for the various groups of mice over time. (C) Proliferation of the cells in tumor section was illustrated by graphing the percentage of Ki67-positive cells in sections of tumor samples. (D) Images of cleaved caspase 3 staining on sections of tumor samples. (E) Tumor pH measurements were performed using mice with tumors treated for one week with the indicated treatment plus one of the three rounds of chemoradiation. (F) The staining of Foxp3(+) cells on tumor samples was plotted as a percentage of Foxp3(+) cells per field.
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Grants and funding

This project was funded by the R01 CA180033-04 entitled: “Molecular mechanisms by which the diabetic drug metformin kills cancer cells” from the National Cancer Institute (NCI), and NIH Center of Biomedical Research Excellence (COBRE) P20GM103548 (NIH).

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