UDCA Suppresses Lung Cancer Cell Proliferation
Our investigation commenced with a rigorous analysis of UDCA's influence on the proliferation dynamics of A549 and H1299 lung cancer cell lines, and the effects were further extrapolated to HUEVC cells (Fig. 1A–C). Through meticulous deployment of CCK-8 assays, we unearthed a pronounced dose-responsive relationship, wherein UDCA concentrations at 1 mmol/L culminated in a reduction of survival rates to 57.12% and 48.2% for A549 and H1299 cells, respectively, while HUEVC cells registered a rate of 64.88%. This discovery not only elucidates the anti-proliferative properties of UDCA but also highlights its potential biocompatibility and safety—qualities essential in therapeutics.
To supplement these insights with quantitative precision, we employed Annexin V and PI staining assays, embarking on a granular examination of cell viability (Fig. 1D). Categorizing the populations into distinct cellular states—including live, early apoptotic, late apoptotic, and necrotic cells—we unveiled the capacity of even nominal UDCA concentrations to detrimentally impact the viability of both A549 and H1299 lung cancer cells. At elevated UDCA concentrations, this effect was accentuated, with survival rates plummeting to 60.5% for A549 cells (Fig. 1E) and 62.27% for H1299 cells (Fig. 1F), thereby underscoring the potency and potential therapeutic relevance of UDCA in lung cancer intervention.
Corroborating these preliminary findings, we engaged in colony formation assays, yielding a consistent pattern of dose-dependent inhibition (Fig. 1G–I). The manifestation of a marked decrement in crystal violet-stained cell colonies, concomitant with escalating UDCA concentrations, resonated strongly with our initial observations, lending further credence to UDCA's robust anti-proliferative capabilities.
UDCA Enhances Chemotherapy Efficacy and Inhibits DOX-Induced Autophagy
Building upon our foundational insights into UDCA's impact on cell proliferation, we next embarked on an incisive exploration of UDCA's potential to augment the therapeutic efficacy of DOX in the context of two human-derived non-small cell lung cancer cell lines, A549 and H1299. Our experimental design encompassed four disparate groups, each reflecting a unique therapeutic modality: Control, UDCA (0.5mmol/L), DOX (2µmol/L), and a synergistic ensemble of UDCA (0.5mmol/L) + DOX (2µmol/L).
Leveraging the precision of the CCK8 assay to scrutinize cell viability, we discerned that while both UDCA and DOX, as singular entities, indeed wielded inhibitory influence over tumor cell proliferation, their combined orchestration was profoundly more efficacious (Fig. 2A, B). This dual treatment modality yielded a suppression of tumor cell growth that was not merely additive but markedly synergistic.
This intriguing synergy was further corroborated through flow cytometric analysis, deploying Annexin V/Propidium Iodide (PI) staining. The data unveiled an unambiguous cytotoxic effect attributable to both UDCA and DOX independently; however, their conjoint administration demonstrably eclipsed the effects of either singular regimen (Fig. 2C–E). The potency of this synergistic relationship between UDCA and DOX thus emerged as a salient theme.
The notion of synergism was buttressed further through the utilization of a colony formation assay to assay the clonogenic capabilities of the targeted cell lines. Where the control group flaunted robust clonogenic resilience, the interventions with either UDCA or DOX disrupted this clonogenicity, and their amalgamation most decisively quashed it (Fig. 2F–H).
Venturing into the complex terrain of autophagy—a known confounder in lung cancer cells' resistance to DOX—we employed the Cyto-ID stain kit to illuminate the landscape of autophagic vesicle emergence under our manifold treatment conditions. A stark contrast was drawn between the minimal fluorescence in the control group and the conspicuous proliferation of green fluorescent spots within the DOX group, the latter indicative of DOX-induced autophagy. Yet, in the combination group, this autophagic response was significantly quelled (Fig. 2I, J). This led us to hypothesize that UDCA may have the potential to neutralize DOX-triggered autophagy within non-small cell lung cancer, effectively dismantling a key tolerance mechanism. The implications of this observation extend beyond the immediate results, laying a substantive foundation for subsequent inquiries into the intricate dance between UDCA, DOX, and autophagy, as these actors converge on the stage of non-small cell lung cancer therapy.
Autophagy Manipulation with UDCA in Cancer Cells
Beclin1, a luminary within the intricate orchestration of cell biology, commands recognition as the inaugural key element discovered in the autophagy pathway[27, 28]. Its imperative function in choreographing autophagosome genesis and accentuating autophagy is incontrovertible. Alongside Beclin1, P62 (or SQSTM1)[29], a versatile cargo protein, graces a myriad of cells and tissues with its presence, where it assumes a pivotal role in bridging LC3 and ubiquitinated substrates, ultimately integrating into autophagosomes and submitting to a designated degradation sequence within autolysosomes[30, 31].
This ballet of autophagy begins with the rendezvous of autophagosomes and lysosomes, precipitating the enzymatic decomposition of proteins or organelles ensconced within autophagic vesicles, epitomized by the degradation of p62[32]. This metamorphosis concludes in the discernible diminution of p62 levels. In juxtaposition, the constriction of autophagy incites a cascade culminating in autophagosome proliferation and a parallel ascent in p62 levels[33, 34]. Within this multifaceted flux, the transmutation of LC3-I to its lipidated analog, LC3-II, demarcates a unique trail leading to the binding of p62 and its ensuing assimilation into autophagosomes[35]. Consequently, the LC3-II/LC3-I ratio crystallizes into a tangible measure of autophagic intensity[36, 37].
In our investigative odyssey, we selected a trio of paramount proteins in autophagy—Beclin1, P62, and LC3B—and leveraged the precision of Western blotting to quantify the nuanced modifications in autophagic propensity induced by a spectrum of UDCA concentrations. As elucidated in Fig. 3A-D, our data unambiguously delineate an inverse dynamic between UDCA concentration and Beclin1 expression, harmoniously synchronized with a proportional decline in the LC3B II/LC3B I ratio. Antithetically, P62 expression levels were observed to resonate directly with escalating UDCA concentration.
Advancing our inquiry, we dissected the faculty for autophagic vesicle genesis under the tutelage of specific drug titrations. Our observations, etched into Fig. 3E, F, resonated with the unmistakable conclusion that UDCA, within the domain of 0.5mmol/L and 1mmol/L, markedly curtailed the emergence of cyto-ID spots. This salient revelation not only amplifies our understanding of UDCA's intricate modulation of autophagy but also augments the burgeoning corpus of evidence that anoints UDCA as a promising harbinger of innovative therapeutic horizons.
UDCA Suppresses Autophagy via TGF-β Inhibition
As our foregoing inquiries illuminated the contours of UDCA's orchestration within the theatre of autophagy, the nuanced tapestry weaving these mechanistic intricacies remained enshrouded in mystery. In this domain, Transforming Growth Factor Beta (TGF-β), a symphony conductor of multifaceted cellular dynamics—spanning cell growth, differentiation, apoptosis, and immune orchestration—assumes a starring role[38, 39]. Within the cellular ballet, TGF-β's elegance extends to choreographing the autophagic process. A body of scholarly artistry attests to the notion that a crescendo in TGF-β levels resonates as a potent enhancer of tumor cell autophagy[40].
Embarking on a scholarly odyssey to map the intricate pas de deux between UDCA and TGF-β, we wielded the refined instrument of ELISA to chronicle the expressions of TGF-β1, TGF-β2, and TGF-β3 within the supernatant of A549 and H1299 cells (Fig. 4A, B), subsequent to their serenade by either UDCA or DOX. Unveiling the score of our findings, the spotlight fell upon DOX, which maintained a studied poise, leaving TGF-β1 levels unaltered, while UDCA, in the key of 0.5mmol/L, resonated with a striking diminuendo in TGF-β1 production. The ensemble of combined treatments further crescendoed to a remarkable waning in TGF-β1.
To fortify these resonant discoveries, we summoned the artistry of both PCR (Fig. 4C, D) and Western blotting techniques (Fig. 4E-H), sculpting an investigative fresco into the nuanced variations in TGF-β1's mRNA cadence and protein expression across the diverse repertoires of treatment. These brushstrokes, harmonizing with the ELISA etching, brought into vivid relief UDCA's pronounced suppressive leitmotif on TGF-β1's expression. This magnum opus not only underscores the hypothesis that UDCA's adagio in autophagy may be inseparably intertwined with its ability to mute TGF-β's expression but also contributes a rich texture to the current scholarly canvas. Our work thus unveils an elegant landscape, resonating with the promise of novel and targeted overtures in the concert hall of cancer therapeutics.
UDCA inhibits TGF-β-induced MAPK phosphorylation in lung cancer cells
Previous research has unequivocally illuminated the pivotal role of non-Smad signaling pathways, specifically the mitogen-activated protein kinase (MAPK) pathway—a sophisticated triptych comprising extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and the p38 MAPK cascade—in the orchestration of autophagy, often guided by the hand of transforming growth factor beta (TGF-β)[41, 42]. With this foundation, our investigation sought to delve into the shadowed intricacies of UDCA's potential regulatory influence within this MAPK symphony, employing Western blotting to scrutinize UDCA's effect on the phosphorylation of ERK and JNK[43, 44].
Outlined in the harmonic structure of Fig. 5A-C, the ensemble of UDCA at a concentration of 0.5mmol/L orchestrated a significant diminuendo in the phosphorylation of both ERK and JNK, akin to a masterful conductor's pause. Yet, this
pause rang hollow with the entrance of TGF-β, which negated UDCA's inhibitory silence. An intricate visual soliloquy provided by Cyto-ID staining, further unfolded, revealing that UDCA's suppressive whisper on A549 cell autophagy was silenced by the crescendo of TGF-β's presence (Fig. 5D, E).
Converging like thematic strains within a symphonic work, our findings compose a hypothesis resonating with insight: UDCA's inhibitory aria on autophagy within the theater of non-small cell lung cancer cells, as induced by the dramatic overture of DOX, may primarily be a masterful orchestration through the modulation of the TGF-β-MAPK pathway. This harmonic conjecture, a motif running through our experimental score, finds echoes in the poignant observation that standalone DOX exposure crescendos the phosphorylation of ERK and JNK, whereas the duet of UDCA plays a counterpoint, tempering the activation within the MAPK serenade (Fig. 5F-H).
Combined Effects of UDCA and DOX in Inhibiting Tumor Growth In Vivo
To explore the in vivo symphony of UDCA's influence on DOX efficacy, we orchestrated a subcutaneous xenograft tumor model, utilizing A549 cells as instrumental performers within the living theater of BALB/c nude mice. Upon the successful crescendo of tumor formation (V = 100mm^3), our ensemble of experimental subjects was divided into four distinct movements, each representing a unique treatment group (Fig. 6A). Echoing our in vitro overtures, both UDCA and DOX conducted individual diminuendos in tumor growth. Yet, the masterful duet of UDCA and DOX composed a significant coda, not only inhibiting the tumor's relentless progression (Fig. 6B, C, F-I) but also extending the survival duration (Fig. 6D) of the treated mice—without crescendoing DOX-induced toxicity. The culmination of our observations was eloquently underscored by the coherent fluorescent duality of Tunnel and caspase 3 staining, complemented by H&E histological examination in the procured tumor samples (Fig. 6O). This pronounced apoptotic manifestation, especially evident in the combined treatment group, stood in stark contrast to the negligible apoptosis in the control group and the comparatively limited apoptosis induced by either UDCA or DOX alone.
C, F-I. Growth curves of mouse tumors. D. Changes in mouse body weight over time. E. Survival curve. J. Immunofluorescence staining to detect TGF-β expression in tumor tissues. K. Western Blot analysis of autophagy in tumor tissues. L-N. Quantitative analysis of Beclin-1, LC3B, and P62.O. H&E staining of mouse tumor tissues and fluorescence dual staining of caspase3/tunnel.
Our scientific score turned to the visual aria of immunofluorescent staining, choreographing a dance to analyze the post-treatment expression of TGF-β within the tumor tissues (Fig. 6J). The lone serenade of DOX cast a vibrant green fluorescence, a luminescent emblem of TGF-β's presence. This vivid illumination was subdued to a gentle pianissimo through the counterpoint of UDCA co-treatment. Western blot's analytical harmony further sang of the tumor's resilience, revealing its ability to mitigate DOX's dissonant attack through autophagy, thus preserving its viability (Fig. 6K-N). The interlude of UDCA, however, interfered with this adaptive dance, lending credence to our resonant hypothesis: UDCA's presence may compose an inhibitory motif against autophagy by conducting a reduction in TGF-β activity, thereby amplifying the cytotoxic crescendo of DOX against the relentless march of non-small cell lung cancer cells.