Pyrroloquinoline Quinone Is an Effective Senomorphic Agent to Target the Pro-Inflammatory Phenotype of Senescent Cells

Abstract

Cellular senescence is an aging-related mechanism characterized by cell cycle arrest, macromolecular alterations, and a senescence-associated secretory phenotype (SASP). Recent preclinical trials established that senolytic drugs, which target survival mechanisms of senescent cells, can effectively intervene in age-related pathologies. In contrast, senomorphic agents inhibiting SASP expression while preserving the survival of senescent cells have received relatively less attention, with potential benefits hitherto underexplored. By revisiting a previously screened natural product library, which enabled the discovery of procyanidin C1 (PCC1), we noticed pyrroloquinoline quinone (PQQ), a redox cofactor that displayed remarkable potential in serving as a senomorphic agent. In vitro data suggested that PQQ downregulated the full spectrum expression of the SASP, a capacity observed in several stromal cell lines. Proteomics data supported that PQQ directly targets the intracellular protein HSPA8, interference with which disturbs downstream signaling and expression of the SASP. PQQ restrains cancer cell malignancy conferred by senescent stromal cells in culture while reducing drug resistance when combined with chemotherapy in anticancer regimens. In preclinical trials, PQQ alleviates pathological symptoms by preventing organ degeneration in naturally aged mice while reserving senescent cells in the tissue microenvironment. Together, our study supports the feasibility of exploiting a redox-active quinone molecule with senomorphic capacity to achieve geroprotective effects by modulating the SASP, thus providing proof-of-concept evidence for future exploration of natural antioxidant agents to delay aging and ameliorate age-related conditions. Prospective efforts are warranted to determine long-term outcomes and the potential of PQQ for the intervention of geriatric syndromes in clinical settings.

Keywords: SASP; age‐related pathologies; aging; cellular senescence; pyrroloquinoline quinone; senomorphics.

FIGURE 1

In vitro screening for senotherapeutic candidates with a natural medicinal agent (NMA) library. (a) A schematic diagram of a stromal cell‐based screening strategy for an NMA library composed mainly of 46 naturally derived agents. Upon completion of the 1st round of senolytics screening, all agents were subjected to the 2nd round of senomorphics screening. The senotherapeutic potential of candidate agents was then validated in several stromal cell lines. (b) Assessment of the effects of individual NMA agents (group A, 21) on the expression of IL‐1α, a core SASP factor, in CTRL, SEN, and SEN cells treated with various agents in culture. (c) Evaluation of the effects of individual NMA agents (group B, 20) on the expression of IL‐1α in CTRL, SEN, and SEN cells treated with various agents in culture. In (b) and (c), each agent was applied at 1 μg/mL. CTRL, control. SEN, senescent. Data in (b) and (c) are shown as mean ± SD and representative of three independent biological replicates, with p values calculated by Student's two‐sided t‐tests. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

Examination of PQQ effects on cellular senescence and expression profile of senescent cells. (a) Cellular senescence determination by SA‐β‐gal staining. Left, representative images of SA‐β‐gal staining. Right, comparative statistics. Scale bar, 20 μm. (b) Cell cycle arrest evaluation by EdU staining. Left, representative images of EdU staining. Right, comparative statistics. Scale bar, 15 μm. (c) Quantitative transcriptional analysis of core SASP factor expression upon BLEO‐induced senescence in the absence or presence of PQQ applied at increasing concentrations. (d) Heatmap displaying the expression landscape of human stromal cells (PSC27) upon senescence and/or PQQ intervention. Human genes were ordered and clustered according to fold change of upregulation in CTRL versus SEN cells and their functional relevance, with corresponding changes upon PQQ treatment exhibited side‐by‐side. PQQ was applied at a concentration of 100 μM. (e) GSEA output presentation of the enrichment of a significant gene set indicative of the development of a typical SASP. (f–h) Pie charts displaying the biological process (f), molecular function (g) of cellular component (h) of top 100 genes that were most downregulated upon treatment by PQQ in senescent PSC27 cells. For all datasets, cells were collected for analyses 7–8 days after senescence induction by BLEO or 3 days after PQQ treatment of senescent cells in culture condition. Data in (a–c) are shown as mean ± SD and representative of three independent biological replicates. p values calculated by two‐sided t‐tests. ^p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 3

Characterization of the molecular mechanism supporting PQQ to curtail SASP expression in senescent cells. (a) Immunoblot assay of several key factors of functional relevance to regulate the SASP development. Senescence of PSC27 cells was induced by treatment with BLEO. GAPDH, loading control. (b) Volcano plot displaying the significantly differentially regulated proteins (Red, upregulated; Blue, downregulated) in the DARTS assay. Senescent stromal cell lysis solution was treated with DMSO (CTRL) or PQQ. (c) A clustering heatmap that profiles proteomic expression change as depicted in (b). Red stars denote PQQ target proteins that may play essential roles in mediating its effect on senescent cells. (d) CETSA assay evaluation of the thermal stabilization of HSPA8 upon incubation with PQQ at a gradient range of temperatures from 30°C to 70°C in protein lysates of PSC27 cells. (e) SPR demonstration that HSPA8 directly binds to PQQ. In the SPR assay, HSPA8 was treated with PQQ over a range of concentrations from 3.25 to 125 μM. KD value is shown beside the traces. SPR, surface plasmon resonance. (f) In silico molecular modeling of PQQ bound to Glu268 and Asp366 of HSPA8. Reported X‐ray diffraction microscopy structure (PDB ID: 6ZYJ) was applied to perform molecular docking, with Glu268 and Asp366 highlighted in the structure. The structure of PQQ is provided at the right, with residues that potentially interact with HSPA8 illustrated. Docking between HSPA8 and PQQ: S = −6.360 kcal/mol. (g) Quantitative PCR measurement of SASP expression upon various treatments of cells as indicated. Data in (g) are shown as mean ± SD and representative of three independent biological replicates. p values were calculated by two‐sided t‐tests. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 4

PQQ reduces the malignancy of prostate cancer (PCa) cells conferred by senescent stromal cell‐derived conditioned media. (a–f) PCa cell lines (PC3, M12, DU145, and LNCaP) were cultured for 3 days with conditioned media (CM) from PSC27 stromal sublines and subjected to assays for proliferation (a), migration (b–d) and invasion (e, f). The CM was collected from an equal number of cells per condition, with a starting DMEM containing 0.5% FBS to make the CM. Wound healing (c) and crystal violet (d) assays were performed to evaluate the migration capacity of cancer cells, with statistical analysis performed to measure (d). (e, f) Invasion assay to examine the invasiveness of cancer cells, with statistical analysis (e) and representative images (f) shown. (g) Chemoresistance assay of PCa cells cultured with the CM from PSC27 sublines described in (a). Mitoxantrone (MIT) was applied at the concentration of IC50 value predetermined per PCa line. (h) Dose–response curves (nonlinear regression/curve fit) plotted from MIT‐based survival assays of PC3 cells cultured with the CM of native PSC27 or BLEO‐induced senescent cells (PSC27‐BLEO) and concurrently treated by a range of concentrations of PQQ. In (c), (d), and (f), scale bars, 50 μm. Data are representative of 3 independent experiments. All p values were calculated by two‐sided t‐tests. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 5

Combination of chemotherapy and PQQ improves therapeutic outcomes in preclinical trials. (a) Schematic representation of the experimental design in severe combined immunodeficient (SCID) mice. Two weeks after subcutaneous implantation of tissue recombinants, animals received either single‐agent or combination therapy administered in metronomic cycles. (b) Statistical comparison of tumor end volumes between treatment groups. PC3 cells were xenografted either alone or together with PSC27 cells to the hind flank of SCID mice, with MIT administered to induce tumor regression. Left, comparative statistic. Right, representative tumor images. The chemotherapeutic agent mitoxantrone (MIT) was used to induce tumor regression. (c) Quantitative measurement of the expression of SASP factors IL‐6, CXCL8, IL‐1α, and AREG at the transcription level. Tissues from animals implanted with both stromal and cancer cells were subject to laser capture microdissection (LCM) isolation, total RNA preparation, and expression assays. (d) Expression analysis of senescence markers including p16^INK4a and p21^CIP1 with tissues from animals as described in (c). (e) Determination of cellular senescence in tumor tissues. Left, representative images of SA‐β‐gal staining in xenografts of each group. Scale bar, 200 μm. Right, comparative statistics. (f, g) Statistical appraisal of DNA damage and apoptosis in the tumor specimens analyzed in (e). Values are presented as a percentage of cells positively stained by immunofluorescence staining with antibodies against γH2AX or caspase 3 (cleaved). Biopsies of placebo‐treated animals served as negative controls for MIT‐treated mice. Scale bars, 50 μm. Data in (b–g) are representative of three independent experiments. All p values were calculated by Student's t‐tests. ^p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 6

PQQ alleviates age‐related pathological changes in solid organs of naturally aged mice without affecting tissue‐level senescence. (a) Schematic representation of treatment schedule for naturally aged mice. (b–d) Quantitative measurement of SASP expression at transcription level in liver, kidney and spleen, tissues. (e) IHC analysis of F4/80 expression in liver tissues. Left, representative images of F4/80 staining in xenografts of each group. Scale bar, 20 μm. Right, comparative statistics. (f) Masson's trichrome staining of kidney tissue. Left, representative images of Masson's trichrome staining in xenografts of each group. Arrows, collagen fibers. Scale bar, 20 μm. Right, comparative statistics. (g) Hematoxylin and eosin (H&E) staining was performed on tissue sections from liver, kidney, and spleen to evaluate pathological features associated with aging. Representative images show the morphology of each organ, highlighting the characteristic alterations indicative of aging, such as tissue degeneration and fibrosis. Arrows in liver and kidney denote areas that exhibit tissue degeneration. Arrows in spleen denote boundaries between the red and white pulp. Scale bar, 50 μm. Data are shown as mean ± SD and representative of three independent experiments (b–f). p values were calculated by Student's t‐test. ^p > 0.05. *p < 0.05. **p < 0.01; ***p < 0.001; ****p < 0.0001.