Abstract

   Lung cancer is a leading cause of cancer-related deaths globally, with
   current treatments having significant limitations, including drug
   resistance, metastasis, and tumor heterogeneity. This study
   investigated the anticancer potential of isalpinin, a flavonoid
   isolated from Paphiopedilum dianthum, against non-small cell lung
   cancer (NSCLC) cell lines A549, H23, and H460. Isalpinin significantly
   inhibited NSCLC cell viability in a dose- and time-dependent manner;
   H23 and H460 cells showed greater sensitivity (IC[50] a ~ 44 μM at 48
   h) compared to A549 cells (IC[50] 82 μM). Isalpinin suppressed
   proliferation, migration, and anchorage-independent growth,
   particularly in H23/H460 cells. Mechanistically, it induced apoptosis
   via increased ROS production and Bcl-2 downregulation, particularly in
   H23 and H460 cells. In a molecular docking analysis, isalpinin was
   found to directly bind to the ATP-binding pocket of AKT1, as confirmed
   by reduced Akt/GSK3β phosphorylation. These results suggest that
   isalpinin showed a potent multi-target natural compound against NSCLC
   that disrupts the key hallmarks of malignancy and pro-survival
   signaling. However, its subtype-specific efficacy warrants further in
   vivo studies and an investigation of combinatorial therapeutic
   approaches to elucidate its clinical potential.

   Keywords: Paphiopedilum dianthum, isalpinin, flavonoid,
   anti-proliferation, anti-migration, anchorage-independent growth,
   non-small lung cancer cells

1. Introduction

   Lung cancer remains one of the most significant global health burdens
   and the leading cause of cancer-related mortality worldwide [[36]1].
   Although its incidence and outcomes vary geographically largely due to
   differences in smoking prevalence, environmental exposures, and
   healthcare access, the disease continues to account for a substantial
   proportion of cancer-related deaths [[37]2,[38]3]. Tobacco smoking is
   the primary risk factor, but other contributors include occupational
   and environmental carcinogens (e.g., radon, asbestos), genetic
   predispositions, and chronic pulmonary conditions [[39]4]. Lung cancer
   is broadly categorized into non-small cell lung cancer (NSCLC) and
   small cell lung cancer (SCLC), each with distinct pathological features
   and treatment challenges [[40]5]. NSCLC comprises approximately 85% of
   all lung cancer cases and includes adenocarcinoma, squamous cell
   carcinoma, and large cell carcinoma. Although NSCLC tends to progress
   more slowly than SCLC, it is often less responsive to conventional
   therapies. In contrast, SCLC is associated with rapid growth and early
   metastasis and requires aggressive therapeutic approaches [[41]6].
   Despite advancements in molecular-targeted therapies and immunotherapy,
   major limitations such as drug resistance to EGFR tyrosine kinase
   inhibitors which often arise from secondary mutations like T790M
   [[42]7], adverse effects, and the influence of the tumor
   microenvironment continue to impede successful long-term outcomes
   [[43]8].

   The phosphoinositide 3-kinase (PI3K)/AKT pathway plays a pivotal role
   in the critical signaling cascades involved in lung tumorigenesis. This
   pathway regulates cellular processes including growth, survival,
   metabolism, and motility. The hyperactivation of Akt, particularly
   through phosphorylation at Ser473, is associated with a poor prognosis
   and therapeutic resistance in NSCLC. One of its downstream targets,
   glycogen synthase kinase 3 beta (GSK3β), is inactivated by
   phosphorylation at Ser9, which contributes to tumor progression.
   Therefore, modulation of this pathway has emerged as a promising
   strategy for the development of anticancer drugs.

   Flavonoids, a diverse group of naturally occurring polyphenols found in
   plants, have gained substantial attention for their potential in cancer
   chemoprevention and therapy due to their anti-inflammatory,
   antioxidant, and antineoplastic activities [[44]9]. These compounds are
   known to interact with various oncogenic pathways including the
   PI3K/Akt and MAPK/ERK pathways. For example, eupafolin, derived from
   Salvia plebeia, was reported to inhibit the proliferation, migration,
   and invasion of NSCLC cells by modulating FAK/PI3K/Akt signaling and
   downregulating metastasis-associated markers, such as MMP9 and RhoA
   [[45]10]; kaempferol derivatives and platanoside from Angelica
   acutiloba induce pyroptosis in A549 cells through the NF-κB/NLRP3
   inflammasome pathway [[46]11].

   Despite the growing body of evidence supporting the anticancer
   potential of flavonoids, the specific effects and mechanisms of
   isalpinin, a flavonol isolated from Paphiopedilum dianthum
   (Orchidaceae), in NSCLC remain unexplored. The Orchidaceae family is
   recognized as a rich source of bioactive compounds with diverse
   pharmacological activities, including significant anticancer
   properties. A recent review by [[47]12] highlights that orchid species
   such as Dendrobium, Bulbophyllum, and Anoectochilus produce
   phenanthrenes, bibenzyls, and flavonoids with cytotoxic effects against
   various cancer cell lines, including lung, breast, and liver cancers.
   These compounds induce apoptosis, cause cell cycle arrest, and modulate
   pathways like PI3K/Akt and caspase activation. Notably, dendrobine from
   Dendrobium nobile and compounds from Anoectochilus roxburghii trigger
   apoptosis in tumor cells. The review also underscores the need for
   further research on the anticancer potential of lesser studied orchid
   species. P. dianthum is an orchid species native to parts of Southeast
   Asia, including northern Thailand. Phytochemical analyses of its roots
   have identified isalpinin ([48]Figure 1) as a key constituent,
   structurally characterized as 5-hydroxy-7-methoxyflavonol based on
   spectroscopic studies [[49]13,[50]14]. Notably, isalpinin lacks proton
   signals at C-2 and C-3, supporting its classification as a flavonol.
   While its effects on non-small cell lung cancer (NSCLC) cell lines have
   not been previously reported, the primary focus of this study isalpinin
   has shown selective cytotoxicity against the doxorubicin-resistant
   breast cancer cell line MCF-7/DOX (IC[50] = 76.24 ± 1.46 μM), without
   affecting normal NIH/3T3 fibroblasts [[51]15]. This selectivity may be
   linked to the presence of a methoxy group at C-7, structurally similar
   to pinosylvin monomethyl ether, which could influence drug resistance
   mechanisms. Additionally, isalpinin has been identified as a substrate
   for breast cancer resistance protein (BCRP), though it does not
   significantly modulate MRP1 activity [[52]15]. These observations
   underscore the importance of flavonoid structure activity relationships
   in determining transporter specificity. Given the global burden of
   NSCLC and the role of flavonoids in modulating critical oncogenic
   pathways such as PI3K/Akt, this study aims to investigate isalpinin’s
   potential as a novel therapeutic agent for NSCLC. This study evaluated
   anticancer potential of isalpinin in NSCLC by targeting five hallmarks
   of malignancy: cell survival (cytotoxicity, MTT), proliferation
   (low-density MTT), metastasis (wound healing), tumorigenicity (soft
   agar), and apoptosis (Hoechst 33342 staining/Bcl-2 expression). These
   assays, selected to reflect NSCLC progression mechanisms, collectively
   demonstrated multi-target activity of isalpinin against survival and
   metastatic pathways. Mechanistic studies linked its efficacy to
   PI3K/Akt/GSK3β inhibition, with heightened effects observed in
   aggressive H23 and H460 subtypes. This supports its potential as a
   selective therapeutic for advanced NSCLC.

Figure 1.

   [53]Figure 1
   [54]Open in a new tab

   Chemical structure of isalpinin.

2. Results

2.1. Cytotoxicity of Isalpinin and Cisplatin on NSCLC Cell Lines

   To further investigate its anticancer potential, we assessed the
   cytotoxic potential of isalpinin (0–200 μM) or cisplatin (0–250 μM) for
   24 and 48 h. Cell viability was determined using the MTT assay, and
   IC[50] values were calculated via nonlinear regression analysis
   ([55]Figure 2A–D) and * are summarized in [56]Table 1. The results
   indicate that isalpinin reduced cell viability in a time- and
   dose-dependent manner across all three NSCLC cell lines. After 48 h of
   treatment, H23 and H460 cells showed greater sensitivity, with IC[50]
   values of 44.34 ± 16.34 μM and 44.46 ± 13.40 μM, respectively, compared
   to 81.88 ± 23.36 μM in A549 cells. Notably, isalpinin exhibited minimal
   cytotoxicity toward normal mouse embryonic fibroblast cells (NIH/3T3),
   with IC[50] values exceeding 100 μM. In contrast, cisplatin
   demonstrated significantly higher cytotoxic potency in all tested cell
   lines, with IC[50] values of 19.7 ± 3.34 μM (A549), 33.39 ± 2.95 μM
   (H23), 41.43 ± 8.58 μM (H460), and 14.65 ± 2.58 μM (NIH/3T3). While
   isalpinin was less potent than cisplatin, its moderate cytotoxicity and
   preferential activity against H23 and H460 cells suggest promising
   therapeutic potential, particularly as a lead compound for the
   development of selective anticancer agents.

Figure 2.

   [57]Figure 2
   [58]Open in a new tab

   Cytotoxic effects of isalpinin and cisplatin in A549, H23, and H460
   lung cancer cells. Cells were treated with various concentrations of
   isalpinin (0–200 μM) for 24 (A) and 48 h. (B) or with cisplatin (0–250
   μM) for 24 h (C) and 48 h (D). Cell viability was assessed using the
   MTT assay and expressed as a percentage relative to the untreated
   control group. Data are presented as mean ± SEM from three independent
   experiments (n = 3). Dose-response curves were generated by nonlinear
   regression of log-transformed compound concentrations. * p < 0.05,
   compared with the control group.

Table 1.

   IC[50] values of isalpinin and cisplatin against NSCLC cell lines and
   mouse fibroblast embryonic cell lines following 24 h and 48 h exposure.
   NSCLC Cell Lines/Normal Mouse Fibroblast Embryonic Cell Lines ^a IC[50]
   of Isalpinin (μM ± SEM) ^a IC[50] of Cisplatin (μM ± SEM)
   24 h 48 h 24 h 48 h
   A549 105.2 ± 14.46 81.88 ± 23.36 48.03 ± 4.71 * 19.17 ± 3.34 *
   H23 100.3 ± 24.39 44.34 ± 16.34 * 86.34 ± 8.91 33.39 ± 2.95 *
   H460 103.1 ± 29.42 44.46 ± 13.40 * 66.55 ± 7.59 41.43 ± 8.58 *
   NIH/3T3 >100 >100 36.25 ± 5.64 14.65 ± 2.58
   [59]Open in a new tab

   ^a Concentration that inhibits cell viability by 50%. Data are
   expressed as the mean ± standard error of the mean (SEM) (n = 3). * p <
   0.05 vs. untreated control cells.

2.2. Anti-Proliferative Effect of Isalpinin on A549, H23 and H460 NSCLC Cell
Lines

   Unregulated cellular proliferation is the hallmark of cancer
   progression [[60]16]. To evaluate the anti-proliferative potential of
   isalpinin, A549, H23, and H460 lung cancer cells were treated with
   increasing concentrations of isalpinin (0–50 μM) for 48 h, and cell
   proliferation was assessed using the MTT assay. As shown in [61]Figure
   3, isalpinin exhibited a dose-dependent inhibitory effect on cell
   proliferation, with a minimal impact observed at concentrations below
   40 μM in A549 cells. In contrast, H23 and H460 cells showed a
   statistically significant reduction in proliferation at 40 μM, with
   marked inhibition evident at 50 μM in all the three cell lines. These
   findings suggest that, while lower concentrations of isalpinin exert
   minimal cytostatic effects, higher doses significantly reduce cell
   viability, particularly in H23 and H460 cells, likely through cytotoxic
   mechanisms.

Figure 3.

   [62]Figure 3
   [63]Open in a new tab

   Anti-proliferative effects of isalpinin in NSCLC cell lines. A549 (A),
   H23 (B), and H460 (C) cells were treated with increasing concentrations
   of isalpinin (0–50 μM) for 48 h. Cell proliferation was evaluated using
   the MTT assay, and the results were normalized to the untreated control
   group. Data are presented as mean ± SEM from three independent
   experiments (n = 3). A concentration- dependent reduction in cell
   proliferation was observed, with statistically significant inhibition
   detected in H23 and H460 cells at concentrations ≥ 40 μM and in all
   three cell lines at 50 μM. * p < 0.05 vs. untreated control.

2.3. The Anti-Migratory Effect of Isalpinin on A549, H23, and H460 NSCLC Cell
Lines

   The anti-migratory effect of isalpinin on A549, H23, and H460 NSCLC
   cell line migration plays a critical role in cancer metastasis. To
   evaluate the anti-migratory potential of isalpinin, a scratch
   wound-healing assay was performed on A549, H23, and H460 NSCLC cell
   lines. Cells were treated with non-cytotoxic concentrations of
   isalpinin (0, 20, 30, and 40 µM) to slightly affect proliferation, and
   wound closure was monitored 24 and 48 h post-scratch ([64]Figure 4A–I).
   Isalpinin inhibited wound closure in a concentration-dependent manner
   in all the three cell lines. The result showed that A549 cells
   ([65]Figure 4A) decreased the relative wound area in untreated control
   cells; the relative wound area decreased from 1.0 (immediately after
   scratch, 0 h) to 0.35 at 48 h, corresponding to a wound-healing rate of
   approximately 0.014 h^−1. In contrast, treatment with 40 µM isalpinin
   significantly attenuated wound closure, with the relative wound area
   decreasing from approximately 1.0 to only 0.75 over the same period.
   This equated to a wound healing rate of approximately 0.006 h^−1 (p <
   0.05), representing an approximate 57% inhibition of cell migration
   relative to the untreated control ([66]Figure 4B,C). In H23 cells
   ([67]Figure 4D), the relative wound area in control conditions
   decreased from approximately 1.0 to 0.35 (healing rate ≈ 0.014 h^−1)
   within 48 h. Treatment with 40 µM isalpinin markedly inhibited this
   process, with the wound area only decreasing from approximately 1.0 to
   0.90, yielding a wound healing rate of approximately 0.002 h^−1. This
   represented approximately 86% inhibition of migration ([68]Figure
   4E,F). Similarly, H460 cells exhibited a reduced migratory capacity
   following isalpinin treatment. Control H460 cells showed a decrease in
   wound area from 1.0 to 0.60 (healing rate approximately 0.019 h^−1)
   over 48 h, ([69]Figure 4G), whereas in the presence of 40 µM isalpinin,
   the wound area decreased from 1.0 to 0.80 (healing rate approximately
   0.003 h^−1), corresponding to an approximate 84% inhibition ([70]Figure
   4H,I). At all time points and concentrations, H23 cells exhibited the
   highest sensitivity to isalpinin, followed by H460 and A549 cells.
   Statistically significant reductions in wound healing were observed in
   H23 and H460 cells at all concentrations and time points, whereas A549
   cells showed significant inhibition only at 40 µM for 24 h. Although
   the wound-healing assay predominantly assesses cell migration, a
   potential minor contribution from altered proliferation rates cannot be
   entirely excluded and warrants further investigation. Collectively,
   these findings underscore the potential of isalpinin as an inhibitor of
   metastatic processes in non-small cell lung cancer, primarily through
   the suppression of cell motility.

Figure 4.

   [71]Figure 4

   [72]Figure 4

   [73]Figure 4
   [74]Open in a new tab

   (A,D,G) Representative images of wound closure in A549, H23, and H460
   cells treated with isalpinin (0, 20, 30, and 40 µM) for 0, 24, and 48
   h. (B,E,H) Quantification of the relative wound area (μm^2/μm^2) at
   each time point demonstrates dose- and time-dependent inhibition of
   migration by isalpinin. (C,F,I) Wound healing rates (h^−1) were
   calculated using linear regression from 0 h to 48 h. Treatment with
   isalpinin at ≥30 µM significantly reduced wound closure compared to
   untreated controls in all cell lines. Data represent the mean ± SEM of
   three independent experiments. * p < 0.05, vs. untreated controls.

2.4. Isalpinin Inhibited Anchorage-Independent Growth in A549, H23, and H460
NSCLC Cell Lines

   To assess the long-term anti-tumorigenic potential of isalpinin, a soft
   agar colony formation assay was performed to evaluate its effect on
   anchorage-independent growth, a hallmark of cellular transformation.
   A549, H23, and H460 cells were treated with noncytotoxic concentrations
   of isalpinin (0–40 μM) for 14 days. As shown in [75]Figure 5A,
   isalpinin treatment markedly reduced colony formation in a
   dose-dependent manner in all three cell lines. Quantitative analysis
   demonstrated a significant decrease in colony number at 40 μM
   isalpinin, with reductions of approximately 67% in A549, 56% in H23,
   and 42% in H460 cells, respectively, compared to the untreated control
   ([76]Figure 5B). Similarly, the colony size was significantly reduced
   by approximately 33%, 84%, and 85% in A549, H23, and H460 cells,
   respectively ([77]Figure 5C). Notably, in A549 cells, treatment with 20
   μM isalpinin did not significantly (ns) alter the colony size, whereas
   higher concentrations showed clear inhibitory effects (p < 0.05). These
   findings indicate that isalpinin effectively suppresses
   anchorage-independent growth in lung cancer cells, particularly in H23
   and H460 cells, suggesting its potential to impair tumorigenic capacity
   under nonadherent conditions.

Figure 5.

   [78]Figure 5
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   Isalpinin suppresses anchorage-independent growth of NSCLC cell lines.
   (A) Representative images of colony formation in soft agar of A549,
   H23, and H460 cells treated with isalpinin (0–40 µM) for 14 days. (B)
   Quantification of colony numbers is expressed as percentages relative
   to the untreated control group. (C) Quantification of the colony size
   (μm^2) for each treatment group. The data represent the mean ± SEM of
   three independent experiments. * p < 0.05, compared to the untreated
   control; ns: not significant.

2.5. The Effect of Isalpinin on Intracellular Reactive Oxygen Species (ROS)
Levels in A549, H23, and H460 NSCLC Cell Lines

   The effect of isalpinin on intracellular reactive oxygen species (ROS)
   levels in A549, H23, and H460 lung cancer cells and elevated levels of
   intracellular reactive oxygen species (ROS) levels are known to
   contribute to oxidative stress and may play a role in mediating
   anticancer activity. To determine whether isalpinin induced ROS
   generation, A549, H23, and H460 cells were treated with isalpinin at
   concentrations of 20, 30, and 40 μM for 1 h. Hydrogen peroxide
   (H[2]O[2], 600 μM) was used as a positive control. Intracellular ROS
   levels were quantified using the DCFH[2]-DA fluorescence assay. As
   shown in [80]Figure 6A–C, isalpinin induced a concentration-dependent
   increase in ROS production in all cell lines. In A549 cells ([81]Figure
   6A), isalpinin at 30 and 40 μM elevated ROS levels by approximately
   2-fold relative to the untreated controls. A more pronounced effect was
   observed in H23 cells ([82]Figure 6B), where ROS levels increased
   nearly 8-fold at the same concentrations. Similarly, in H460 cells
   ([83]Figure 6C), isalpinin treatment led to a 6-fold increase in ROS
   generation. These findings suggest that isalpinin induces significant
   oxidative stress in lung cancer cells, with H23 and H460 cells showing
   greater sensitivity to isalpinin-mediated ROS accumulation than the
   A549 cells. This differential ROS induction might contribute to the
   cell-specific cytotoxic effects of isalpinin.

Figure 6.

   [84]Figure 6
   [85]Open in a new tab

   Isalpinin induces intracellular ROS generation in NSCLC cell lines.
   A549 (A), H23 (B), and H460 (C) cells were treated with isalpinin (20,
   30, or 40 μM) or hydrogen peroxide (H[2]O[2]; 600 μM) as a positive
   control for 1 h. Intracellular reactive oxygen species (ROS) levels
   were measured using the DCFH[2]-DA fluorescence assay. Data are
   expressed as fold change relative to untreated control cells and are
   presented as the mean ± SEM from three independent experiments (n = 3).
   * p < 0.05, vs. untreated controls.

2.6. Effect of Isalpinin Induces Apoptosis and Modulates Anti-Apoptotic Bcl-2
Protein Expression in NSCLC Cell Lines

   To investigate whether apoptosis contributes to the anticancer effects
   of isalpinin, A549, H23, and H460 lung cancer cells were treated with
   increasing concentrations of isalpinin (0–40 µM) for 24 and 48 h.
   Apoptotic cell death was assessed using Hoechst 33342 nuclear staining,
   followed by fluorescence microscopy. As illustrated in [86]Figure 7A–F,
   isalpinin induced a concentration- and time-dependent increase in
   apoptosis in all three cell lines. A549 cells ([87]Figure 7A,B) showed
   a modest apoptotic response, with approximately 6.2% apoptosis at 40 µM
   after 24 h, but this increased significantly to nearly 50% after 48 h.
   In contrast, H23 cells ([88]Figure 7C,D) exhibited approximately 60.9%
   apoptosis at 40 µM after 24 h, with the levels remaining stable after
   48 h. H460 cells ([89]Figure 7E,F) demonstrated the highest
   sensitivity, with approximately 69.7% apoptosis at 40 µM after 24 h,
   which increased further to approximately 79.8% at 48 h. These findings
   indicated that isalpinin induces apoptosis in lung cancer cells in a
   time- and dose-dependent manner, with H460 cells being the most
   susceptible, followed by H23 and A549 cells.

Figure 7.

   [90]Figure 7

   [91]Figure 7
   [92]Open in a new tab

   Isalpinin induces apoptosis and downregulates anti-apoptotic protein
   Bcl-2 in NSCLC cell lines. (A,C,E) Representative fluorescence
   microscopy images (20× magnification) of Hoechst 33342-stained A549
   (A), H23 (C), and H460 (E) cells after treatment with isalpinin (0–40
   μM) for 24 and 48 h. Apoptotic cells exhibiting chromatin condensation
   and nuclear fragmentation are also shown. (B,D,F) Quantification of
   apoptotic cells in A549 (B), H23 (D), and H460 (F) cell populations,
   presented as a percentage of total cells. (G,H) H23 (G) and H460 (H)
   cells were treated with isalpinin (0–40 μM) for 24 h, and total protein
   lysates were analyzed using Western blotting to evaluate Bcl-2
   expression levels. GAPDH was used as an internal loading control.
   Densitometric analysis of Bcl-2 levels relative to GAPDH is shown,
   expressed as fold change compared to untreated controls. Data are
   presented as the mean ± SEM from three independent experiments (n = 3).
   * p < 0.05 vs. untreated cells.

   To further elucidate the apoptotic mechanism induced by isalpinin, the
   expression levels of the antiapoptotic protein Bcl-2 were examined in
   H23 and H460 lung cancer cells after a 24 h treatment with isalpinin at
   concentrations of 0, 20, 30, and 40 μM. As shown in [93]Figure 7G,H, a
   Western blot analysis revealed a concentration-dependent reduction in
   Bcl-2 protein levels relative to the GAPDH control. Notably, treatment
   with 40 μM isalpinin resulted in a significant decrease in Bcl-2
   expression by approximately 26% in H23 cells ([94]Figure 7G) and 22% in
   H460 cells ([95]Figure 7H), compared to the untreated control group (*
   p < 0.05). These findings suggest that isalpinin may promote apoptosis
   in lung cancer cells, at least in part, through a downregulation of
   Bcl-2.

2.7. Gene Ontology (GO) and KEGG Pathway Enrichment Analysis of
Isalpinin-Associated Targets

   To elucidate the potential molecular mechanisms underlying the effects
   of isalpinin in non-small cell lung cancer (NSCLC), gene ontology (GO)
   framework, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
   enrichment, analyses were performed on the predicted molecular targets
   of isalpinin. GO terms were categorized into biological processes (BP),
   molecular functions (MF), and cellular components (CC). Analysis of the
   biological processes ([96]Figure 8A), ranked by gene count, indicated
   significant enrichment in several key areas. These include the
   regulation of protein phosphorylation, the cellular response to organic
   substances, the regulation of hormone levels, the response to hormones,
   the regulation of response to stimuli, protein phosphorylation itself,
   the regulation of metabolic processes, cellular protein localization,
   the positive regulation of molecular function ([97]Figure 8B), and the
   regulation of signaling pathways. In terms of molecular functions, the
   predicted targets of isalpinin were predominantly associated with
   transferase activity, particularly involving phosphorus-containing
   groups, and catalytic activity. Other significantly enriched molecular
   functions included protein kinase activity, ATP binding, ion binding,
   enzyme binding, hormone receptor binding, transcription factor binding,
   transcription corepressor activity, and steroid hormone receptor
   activity. These findings suggested that isalpinin may mediate its
   effects by modulating a range of enzymatic and binding activities that
   are crucial for cellular functions in NSCLC. An analysis of the
   cellular components revealed that the predicted targets of isalpinin
   were distributed across various subcellular locations. The enriched
   terms included membrane components ([98]Figure 8C), cytoplasm, plasma
   membrane, extracellular region, cytoskeleton, organelles, cytoplasmic
   vesicles, extracellular space, cell junctions and intracellular
   anatomical structures. This broad distribution suggests that the
   molecular targets of isalpinin are located in diverse cellular
   compartments, potentially affecting multiple cellular pathways and
   functions. Furthermore, KEGG pathway analysis ([99]Figure 8D)
   identified a significant enrichment of the predicted targets of
   isalpinin in pathways that are critically involved in oncogenesis.
   Notably, these included the PI3K/Akt signaling pathway, pathways
   related to miRNAs in cancer, general cancer pathways, nitrogen
   metabolism, endocrine resistance, proteoglycans in cancer,
   prostaglandins in cancer, the Ras signaling pathway, arachidonic acid
   metabolism, and pathways associated with EGFR tyrosine kinase inhibitor
   resistance. The prominent enrichment in the PI3K/Akt pathway and other
   cancer-associated signaling cascades suggests that isalpinin exerts its
   anticancer effects by modulating these pivotal oncogenic signaling
   networks in NSCLC. Collectively, these enrichment analyses provided
   valuable insights into the potential molecular landscape influenced by
   isalpinin in NSCLC cells. These findings highlight the potential of
   isalpinin to target multiple cancer-related pathways and processes,
   thereby inhibiting cell migration and exerting broader anticancer
   effects.

Figure 8.

   [100]Figure 8
   [101]Open in a new tab

   Gene Ontology (GO) and KEGG pathway enrichment analysis of
   isalpinin-targeted genes. (A) Biological Process (BP): Top enriched GO
   terms related to cell death, chemical response, the regulation of
   signaling, and apoptosis. (B) Molecular Function (MF): enrichment in
   catalytic activity, ATP binding, protein kinase activity, and
   nucleotide binding. (C) Cellular Component (CC): Targeted proteins are
   predominantly associated with intracellular anatomical structures,
   membranes, and cytoplasmic components. (D) KEGG Pathway Analysis: Major
   enriched pathways include cancer-related signaling pathways, such as
   PI3K/Akt, pathways in cancer, microRNAs in cancer, EGFR-TKI resistance,
   and hormone-related signaling. Gene counts reflected the number of
   target genes associated with each GO term or KEGG pathway. Data were
   analyzed using enrichment tools with a significance threshold of p <
   0.05.

2.8. Construction of the Protein–Protein Interaction (PPI) Network and
Computational Modeling of AKT with Molecular Docking

   To further elucidate the molecular interactions underlying the effect
   of isalpinin, we constructed a protein–protein interaction (PPI)
   network to identify central nodes and key pathways potentially
   modulated by isalpinin in NSCLC cells, and molecular docking studies
   were conducted focusing on AKT, a central regulator in cancer cell
   survival and proliferation. A protein–protein interaction (PPI) network
   was constructed to systematically elucidate the molecular mechanisms by
   which isalpinin exerts its anticancer effects in NSCLC. This network
   was built by mapping 43 predicted cancer-related targets using the
   STRING v11.0 database ([102]Figure 9A), which allowed identification of
   central nodes and key pathways potentially modulated by isalpinin in
   NSCLC cells. The PPI network revealed that AKT1 occupies a central
   node, indicating a high degree of connectivity with other signaling
   proteins, such as EGFR, PI3KR1, and MAPK1. A topological analysis using
   Cytoscape prioritized AKT1 based on degree centrality and betweenness
   metrics, suggesting that it is a key hub protein in the
   isalpinin–target interaction network. The molecular mechanisms
   underlying the anti-NSCLC effects of isalpinin, identified through PPI
   network analysis, were further elucidated through molecular docking
   simulations. These simulations investigated potential binding
   interactions between isalpinin and key target proteins. Based on PPI
   network construction, which identified AKT1 as a central hub protein in
   the isalpinin–NSCLC interaction network, we focused on characterizing
   the binding mechanism of isalpinin with AKT1. Docking simulations were
   conducted using the X-ray crystallographic structure of activated AKT
   (PDB ID: 1O6L) to predict the binding affinity and interaction sites of
   isalpinin (CB-Dock2 with AutoDock Vina). The best binding pose (highest
   negative Vina score) is shown in [103]Figure 9B, illustrating that
   isalpinin fits into the ATP-binding pocket of AKT1 and interacts with
   residues critical for kinase function. Detailed interaction profiling
   ([104]Figure 9C) revealed multiple stabilizing interactions between
   isalpinin and AKT1. Notably, conventional hydrogen bonding occurred
   with Glu236 and Thr292, whereas π-alkyl interactions were observed with
   Phe439, Ala232, and Tyr231, enhancing the affinity of the compound.
   Additional van der Waals interactions with residues such as Ala166,
   Met282, and Leu158 further stabilize the ligand–protein complex. These
   findings suggest that isalpinin may exert anticancer effects, at least
   in part, by targeting AKT1 through direct binding to its catalytic
   domain, thereby interfering with downstream pro-survival signaling
   pathways.

Figure 9.

   [105]Figure 9
   [106]Open in a new tab

   Construction of a protein–protein interaction (PPI) network and
   molecular docking analysis of isalpinin with AKT1. (A) The PPI network
   of predicted isalpinin target proteins was constructed using the STRING
   v11.0 database and visualized using Cytoscape. Nodes represent
   proteins, and edges indicate predicted protein–protein interactions
   with a confidence score ≥ 0.95. AKT1 (highlighted in dark green)
   emerged as a central hub, indicating its potential importance in
   isalpinin-mediated signaling modulation. (B) Molecular docking of
   isalpinin into the active site of AKT1 (PDB ID: 1O6L) using AutoDock
   Vina on the CB-Dock2 platform. The surface representation illustrates
   the binding pocket, with the inset highlighting key interactions
   between isalpinin and the amino acid residues of AKT1 at atomic
   resolution. The residues involved in binding include Glu236, Ala232,
   Phe439, and Thr292. (C) Two-dimensional interaction map of isalpinin
   docked into AKT1, generated using the BIOVIA Discovery Studio
   Visualizer. The ligand interacted with key residues through hydrogen
   bonds (green lines), π-alkyl interactions (purple), van der Waals
   forces (light green), and electrostatic contacts (orange). These
   interactions collectively support the stable binding of isalpinin to
   AKT1’s ATP-binding site.

2.9. Effect of Isalpinin on Akt/GSK3β Pathway in NSCLC Cell Lines

   Following previous analyses of gene pathways and functional enrichment,
   which highlighted the significant involvement of the Akt signaling
   pathway in the action mechanism of isalpinin, further studies were
   undertaken to determine the direct impact of isalpinin on this pathway
   in NSCLC cell lines. To assess the expression and phosphorylation
   levels of crucial proteins in the Akt/GSK3β pathway, H23 and H460 cell
   lines were treated with increasing concentrations of isalpinin (0, 20,
   30, and 40 µM), and the expression levels of Akt, GSK3β, and their
   phosphorylated forms were evaluated via Western blot analysis. In H23
   cells ([107]Figure 10A), treatment with isalpinin led to a
   dose-dependent decrease in phosphorylated Akt (Ser473) levels, while
   total Akt levels remained stable. Quantitative densitometry showed that
   treatment with 30 and 40 µM isalpinin significantly reduced p-Akt
   levels to approximately 73% and 54%, respectively, compared to
   untreated control cells, (p < 0.05; [108]Figure 10B). Concordantly, the
   phosphorylation of GSK3β at Serine 9 (p-GSK3β), a downstream target of
   Akt, was progressively attenuated with increasing concentrations of
   isalpinin. Treatment with 30 and 40 µM of isalpinin reduced p-GSK3β
   levels by approximately 68% and 49%, respectively, compared to the
   control, (p < 0.05; [109]Figure 10C), whereas the total GSK3β
   expression remained constant. A similar inhibitory pattern was observed
   in H460 cells ([110]Figure 10D). Treatment with 40 µM of isalpinin
   significantly decreased p-Akt levels to approximately 69% of control
   values (p < 0.05; [111]Figure 10E), with total Akt levels remaining
   unaffected. Correspondingly, the phosphorylation of GSK3β (Ser9) was
   also inhibited in a dose-dependent manner, exhibiting a significant
   reduction to approximately 52% of control levels at 40 µM of isalpinin
   (p < 0.05), without altering total GSK3β protein expression
   ([112]Figure 10F). These results clearly demonstrate that isalpinin
   downregulates Akt activation and GSK3β phosphorylation in a
   concentration-dependent manner in both the H23 and H460 cell lines. The
   observed suppression of the Akt/GSK3β signaling axis is consistent with
   the pathway-level predictions from the KEGG enrichment analysis, and
   provides mechanistic support for the anti-migratory and
   anti-proliferative effects of isalpinin. These findings underscore the
   therapeutic potential of isalpinin as a modulator of dysregulated
   oncogenic signaling in NSCLC.

Figure 10.

   [113]Figure 10
   [114]Open in a new tab

   Isalpinin inhibits the Akt/GSK3β signaling pathway in H23 and H460
   non-small cell lung cancer (NSCLC) cells. (A) Representative Western
   blot images showing the expression of total Akt, phosphorylated Akt
   (p-Akt), total GSK3β, and phosphorylated GSK3β (p-GSK3β) in H23 cells
   treated with 0, 20, 30, and 40 µM isalpinin for 24 h. GAPDH was used as
   a loading control. (B,C) Densitometric quantification of p-Akt and
   p-GSK3β in H23 cells normalized to GAPDH. (D) Western blot analysis of
   the same proteins in H460 cells under identical conditions. (E,F)
   Densitometric analysis of p-Akt and p-GSK3β in H460 cells. Data are
   presented as the mean ± SEM from three independent experiments. * p <
   0.05, compared to the untreated control group.

3. Discussion

   Cell lung carcinoma is the most common type of lung cancer, and
   metastasis and apoptosis dysregulation are major obstacles that
   contribute to its poor clinical outcome [[115]17]. Therefore,
   therapeutic approaches remain a challenge for anticancer drug
   discovery. Epidemiological evidence has linked fruit and vegetable
   consumption to a reduced cancer risk, which is often attributed to
   bioactive flavonoids. These polyphenols have been shown to exhibit
   diverse anticancer effects in preclinical models. Natural compounds
   from orchid species have been shown to possess anticancer properties
   because they contain up to one hundred types of biologically active
   flavonols [[116]13,[117]15]. Isalpinin, a methylated flavonol
   structurally related to common dietary flavonoids, has potential
   advantages, possibly including enhanced stability or target
   interactions owing to its methyl group. However, its efficacy,
   particularly in lung carcinoma, remains unclear. This study aimed to
   characterize the multifaceted anticancer properties (cancer cell
   viability, proliferation, mitosis, and apoptosis) of isalpinin, a
   methylated flavonol isolated from P. dianthum, against NSCLC cell
   lines, including A549, H23, and H460.

   Isalpinin exhibited a concentration- and time-dependent inhibition of
   cell viability across all tested NSCLC cell lines (A549, H23, and
   H460), as determined by MTT assays. An initial assessment after 24 h of
   treatment revealed comparable sensitivity across the tested lines, with
   calculated half-maximal inhibitory concentrations (IC[50]) of 105.2 ±
   14.46 µM for A549, 103.1 ± 29.42 µM for H460, and 100.3 ± 24.39 µM for
   H23 cells. These initial potencies are consistent with previous reports
   for the non-methylated flavonol quercetin, which exhibited IC[50]
   values of 106.3 µM and 92.2 µM against A549 and H460 cells,
   respectively, after 24 h [[118]18]. Previous research demonstrated that
   isalpinin exhibited notable cytotoxic activity against the MCF-7 breast
   cancer cell line, with an IC[50] value of 52.2  ±  5.9 μM [[119]15],
   underscoring its broad-spectrum anticancer potential across diverse
   malignancies. Although its cytotoxic potency at this early time point
   was initially lower than that of the conventional chemotherapeutic
   agent, cisplatin, prolonged exposure (48 h) resulted in significantly
   enhanced cytotoxicity, particularly in H23 and H460 cells. Indeed,
   extending the exposure duration to 48 h increases IC[50] values to
   44.46 ± 13.40 µM and 44.34 ± 16.34 µM against H460 and H23 cells,
   respectively. In contrast, A549 cells displayed reduced sensitivity
   over the same period, with an IC[50] value of 81.88 ± 23.36 µM. This
   delayed yet sustained cytotoxic response observed, especially in H23
   and H460 cells, may be attributed to the gradual induction of
   intracellular stress and the engagement of apoptotic pathways. These
   findings underscore the differential cytotoxic potential of isalpinin,
   suggesting that its anti-proliferative efficacy may be more pronounced
   in H460 and H23 cells. The observed IC[50] values at 48 h confirm a
   higher susceptibility of H23 and H460 cells to isalpinin-induced
   cytotoxicity compared to A549 cells, indicating cell line-specific
   responses potentially due to differences in genetic background, redox
   balance, or drug transporter expression, in addition to variations in
   underlying signaling pathway activation [[120]9]. According to the
   IC[50] of isalpinin, approximately 40 µM, which may be considered high
   for direct therapeutic applications, strategies to enhance its clinical
   translatability are crucial. For further applications, encapsulating
   isalpinin within nanocarrier systems, such as liposomes, polymeric
   nanoparticles, or micelles, is a promising solution. Such
   nanoformulations can significantly improve the pharmacokinetic profile
   of isalpinin by increasing its solubility, protecting it from premature
   degradation, and extending its circulation half-life. This approach is
   highly relevant, as flavonoids are being actively investigated as
   adjuvants in clinical trials, such as [121]NCT05724329 (dasatinib and
   quercetin). By utilizing targeted delivery systems to achieve a higher
   local concentration at the tumor site, isalpinin can be effectively
   used in synergistic combinations with other anticancer agents,
   unlocking its full therapeutic potential. Consistent with previous
   reports on kaempferol bioactivity, kaempferol showed time-dependent
   cytotoxicity, reaching IC[50] values of 87.3 µM (A549) and 43.7 µM
   (H460) after 72 h [[122]19], while kaemferide (Kaempferol 4′-O-methyl
   ether) showed IC[50] values of 22.5 ± 1.35, 26.2 ± 1.4, and 29.1 ± 1.5
   µM against A549, H23, and H460 cell proliferation (24 h incubation
   time), respectively [[123]20]. Moreover, quercetin (non-methyled
   flavonol) demonstrated inhibitory effects on A549 human lung carcinoma
   cells, with an IC[50] of 72.2 ± 2.3 µM following a 48 h incubation
   period, whereas isorhamnetin (3′-O-methylquercetin) demonstrated a
   2.7-fold increase in potency (IC[50] = 26.6 ± 1.7 µM) relative to
   quercetin, while tamarixetin (4′-O-methylquercetin) showed a 3.7-fold
   increase (IC[50] = 19.6 µM) in A549 cells. This enhanced efficacy is
   linked to structural methylation [[124]21]. Flavonoid methylation is
   known to influence pharmacokinetic properties, potentially enhancing
   bioavailability and metabolic stability compared to non-methylated
   analogs such as quercetin and kaempferol. This hypothesis is supported
   by the finding that other methylated flavonoids, such as tangeretin and
   nobiletin, exhibit significant anti-proliferative activity [[125]22].
   The notably improved activity of the O-methylated flavonol isorhamnetin
   suggests that its potential utility as an anticancer agent for
   non-small cell lung cancer warrants further investigation, as cell
   migration is a fundamental aspect of cancer metastasis, and inhibition
   of this process represents a key therapeutic strategy. In this study,
   we investigated the anti-migratory potential of isalpinin, a methylated
   flavonol, against lung cancer progression using wound-healing assays in
   A549, H23, and H460 cell lines. Our findings demonstrated that
   isalpinin significantly attenuated cell migration in a dose-dependent
   manner in all three cell lines. Notably, A549 cells exhibited the
   lowest sensitivity, with wound closure inhibited by approximately 60%
   after 48 h of treatment with 40 µM isalpinin, whereas significant
   inhibition was observed in H23 and H460 cells. The observed
   anti-migratory effects were consistent with previous reports of related
   flavonols. Myricetin inhibits migration through MMP and EMT pathways
   [[126]23]. Kaempferol suppresses TGF-β1-induced migration/EMT in A549
   cells via the PI3K/Akt1 pathway [[127]24]. Furthermore, methylated
   flavonol kaempferide reduced A549 cell migration by regulating
   ROS-mediated TGF-β signaling, decreasing pro-migratory proteins (e.g.,
   SNAI 1, N-cadherin), and increasing E-cadherin expression [[128]20].
   These examples support the role of flavonols in targeting key cell
   migration and EMT pathways. The wound-healing assay revealed that
   isalpinin potently inhibited NSCLC cell migration in a dose-dependent
   manner ([129]Figure 4). While transwell invasion assays and EMT marker
   quantification would provide additional mechanistic insights, the
   observed two-dimensional migration suppression strongly suggests an
   anti-metastatic potential. Migration and invasion share overlapping
   molecular drivers, including PI3K/Akt signaling, which was effectively
   inhibited in our study ([130]Figure 8 and [131]Figure 9). Akt
   suppression downregulates MMP-9 and upregulates E-cadherin, thereby
   counteracting EMT and invasion. Future studies will explore the effects
   of isalpinin on three-dimensional invasion and EMT markers, such as
   Snail, Twist, and N-cadherin, to fully elucidate its antimetastatic
   mechanisms. The ability of isalpinin to interfere with these critical
   cellular processes underscores its potential therapeutic applications.
   Subsequent research encompassing detailed mechanistic elucidation and
   preclinical in vivo efficacy evaluations is imperative for advancing
   isalpinin for potential clinical applications in lung cancer treatment
   and the demonstrated significant dose-dependent inhibition of
   anchorage-independent growth in A549, H23, and H460 (NSCLC) cell lines,
   as assessed via a 14 day soft agar colony formation assay. At
   concentrations ranging from 20 µM to 40 µM, isalpinin reduced both the
   number and size of colonies across all three lines, although with
   differential sensitivities. H460 cells exhibited the highest
   sensitivity, followed by H23 cells, whereas A549 cells displayed
   moderate sensitivity to isalpinin treatment under these conditions.
   Compared with previous reports, quercetin inhibited A549 colony
   formation by 27%, 49%, and 83% at concentrations of 25, 50, and 100
   μmol/L, respectively, relative to untreated controls. This effect was
   mechanistically associated with the direct inhibition of Aurora B
   kinase, a target highly expressed in these cells, as confirmed by
   reduced sensitivity following Aurora B knockdown [[132]25]. In
   contrast, the O-methylated quercetin metabolite isorhamnetin exhibited
   substantially greater potency than quercetin, significantly suppressing
   A549 colony formation by approximately 32%, 65%, and 82% at markedly
   lower concentrations of 2.5, 5, and 10 μM, respectively [[133]26]. The
   inhibitory action of isorhamnetin involves distinct mechanisms,
   primarily the induction of mitochondrial-dependent apoptosis and the
   modulation of autophagy, where autophagy inhibition enhances apoptotic
   cell death [[134]26]. These flavonoids share several common mechanisms
   in inhibiting colony formation, including the induction of apoptosis
   through the modulation of pro-apoptotic (Bax and Bad) and
   anti-apoptotic (Bcl-2) proteins [[135]27,[136]28,[137]29,[138]30], the
   activation of caspase-dependent pathways leading to cell death
   [[139]27,[140]28,[141]30], the induction of autophagy, which
   contributes to cancer cell death [[142]28,[143]29], the inhibition of
   signaling pathways critical for cancer cell survival and proliferation,
   particularly PI3K/Akt/mTOR [[144]26], and cell cycle arrest at various
   phases, preventing cancer cell division and proliferation
   [[145]27,[146]28,[147]30]. Further research specifically addressing the
   effects of these natural compounds on H460 and H23 cell colony
   formation would expand our understanding of their potential as
   therapeutic agents in lung cancer.

   A549, H23, and H460 cells have been reported to exhibit a differential
   expression of Nrf2/HO-1, a key component of the antioxidant defense
   system, contributing to distinct redox homeostasis mechanisms
   [[148]31]. Therefore, we determined whether isalpinin, a methylated
   flavonol, exerted anticancer effects against ROS generation. A549, H23,
   and H460 cells were treated with isalpinin (20–40 µM) and intracellular
   ROS levels were quantified. Isalpinin elevated ROS levels in a
   concentration-dependent manner in all cell lines. Significant
   differential sensitivity was observed in H23 cells which showed the
   largest response (approximately 8-fold ROS increase vs. control),
   followed by H460 cells (approximately 6-fold), whereas A549 cells
   exhibited a modest (approximately 2-fold) increase. This sensitivity
   pattern contrasts sharply with that of kaempferide, which potently
   induces ROS in A549 cells compared to that in H460 and H23 cells
   (reportedly via mitochondrial electron transport chain disruption)
   [[149]20]. Isalpinin showed lower efficacy in A549 cells but higher
   activity in H23 and H460 cells. The reduced sensitivity of A549 cells
   to isalpinin may be correlated with their characteristically high
   expression of Nrf2/HO-1, a key component of the antioxidant defense
   system known to confer resistance to oxidative stress (e.g., cisplatin
   resistance) [[150]31]. Conversely, H23 cells exhibited defective Nrf2
   activation, and both H23 and H460 cells had lower basal Nrf2/HO-1
   levels, potentially rendering them less capable of mitigating
   isalpinin-induced ROS. While kaempferide effectively overcomes A549
   defenses, possibly through direct mitochondrial targeting,
   isalpinin-induced ROS production appears to be more readily
   counteracted by the robust Nrf2/HO-1 system in A549 cells
   [[151]20,[152]31]. Consistent with ROS levels, isalpinin-induced
   apoptosis was significantly higher and occurred earlier in H23 and H460
   cells than in A549 cells. H460 and H23 cells displayed substantial,
   concentration-dependent apoptosis after 24 h (reaching approximately
   60–70% at 40 µM in H460), which increased further by 48 h. In contrast,
   A549 cells showed minimal apoptosis at 24 h, with significant induction
   only apparent after 48 h (reaching approximately 50% apoptosis at 40
   µM). This direct correlation between the magnitude of ROS generation
   and apoptotic rates underscores the role of oxidative stress in the
   cytotoxicity of isalpinin and highlights the influence of cellular
   antioxidant capacity modulated by Nrf2/HO-1 in determining sensitivity
   [[153]20,[154]31].

   Network-based interactome analysis identified PIK3R1, SRC, and ESR1 as
   highly connected nodes within cancer-associated signaling networks.
   These hubs converge on the PI3K/Akt pathway, a critical regulator of
   NSCLC cell proliferation, survival, and migration [[155]32]. To
   contextualize these findings, we employed three NSCLC cell lines
   well-validated in in vitro models for lung cancer biology and drug
   response. Molecular docking simulations revealed that isalpinin, a
   methylated flavonol, binds directly to the ATP-binding pocket of PIK3R1
   (p85α subunit of PI3K), suggesting its potential as a PI3K/Akt pathway
   inhibitor. Experimental validation in H460 cells confirmed that
   isalpinin treatment reduces AKT (Ser473) and GSK3β (Ser9)
   phosphorylation, consistent with pathway inhibition. The structural
   basis for this interaction lies in the isalpinin planar flavonol
   scaffold, which facilitates π–π stacking with aromatic residues (e.g.,
   Phe439, Tyr231) and hydrogen bonding via hydroxyl/methoxy groups (e.g.,
   Glu236, Thr292). Density functional theory calculations further support
   the coplanarity of rings A, B, and C in methylated flavonols like
   isalpinin, enhancing electron delocalization and binding affinity
   [[156]33,[157]34]. To broaden the mechanistic insights, we integrated
   NSCLC-specific transcriptomic datasets (TCGA LUAD/LUSC cohorts) and
   pathway enrichment analysis (GO/KEGG). These analyses highlighted
   PI3K/Akt signaling as a key dysregulated pathway in NSCLC, aligning
   with isalpinin predicted targets from STRING DB and DrugBank. Flavonols
   have been reported to interact with PI3K/Akt components*, reinforcing
   the relevance of the mechanism of isalpinin in NSCLC pathophysiology
   [[158]35].

   Nuclear morphology assays using Hoechst 33342 staining revealed that
   isalpinin induced apoptosis in all three cell lines in a dose- and
   time-dependent manner, with the highest levels observed in H460 cells,
   followed by H23 and A549 cells. Specifically, H460 cells exhibited
   apoptosis rates approaching 80% after 48 h at 40 µM of isalpinin,
   indicating potent pro-apoptotic activity. A Western blot analysis
   further confirmed the downregulation of Bcl-2, an anti-apoptotic member
   of the Bcl-2 family, in both H23 and H460 cells. The suppression of
   Bcl-2 aligns with the activation of the intrinsic (mitochondrial)
   apoptotic pathway, as Bcl-2 plays a crucial role in maintaining the
   mitochondrial membrane integrity and preventing cytochrome c release.
   Its downregulation likely facilitates the activation of the apoptotic
   cascade via caspase-9 and -3. Supporting this, isorhamnetin was shown
   to inhibit the PI3K/Akt/GSK3β axis in hepatocellular carcinoma. It
   induces apoptosis via mitochondrial pathways by increasing
   pro-apoptotic proteins (Bax and cleaved caspases) and suppressing
   anti-apoptotic Bcl-2 without activating ROS-mediated mechanisms. The
   apoptotic effects were entirely PI3K/Akt dependent, as shown by the
   consistent downregulation of pathway activity and effective suppression
   of tumor growth in vivo [[159]33]. These results contrast with those of
   kaempferide, which exerts anticancer effects via the ROS-dependent
   activation of JNK and the suppression of PI3K/Akt and quercetin, which
   disrupt mitochondrial integrity via ROS accumulation and Sestrin2-AMPK
   activation [[160]20]. While Hoechst staining effectively demonstrated
   characteristic apoptotic morphology, including chromatin condensation
   and nuclear fragmentation, we acknowledge that a direct measurement of
   cleaved caspase-3 would provide additional mechanistic insight into
   apoptotic pathway activation [[161]36]. Hoechst 33342 staining is a
   well-established and reliable method for detecting apoptosis based on
   nuclear morphological changes, which represent fundamental hallmarks of
   early apoptosis [[162]37,[163]38]. In our study, significant Bcl-2
   downregulation ([164]Figure 7G,H), together with the characteristic
   apoptotic morphological changes observed by Hoechst staining
   ([165]Figure 7A,C,E), provide multiple independent lines of evidence
   supporting apoptosis induced by isalpinin. The dose-dependent
   relationship between isalpinin treatment and both morphological changes
   and Bcl-2 expression further supports this conclusion. The observed
   nuclear condensation and fragmentation are consistent with classical
   apoptotic cell death, while concurrent Bcl-2 downregulation indicates
   activation of the mitochondrial-mediated apoptotic pathway.

   The temporal progression of apoptotic events explains the differences
   observed between Hoechst fluorescence at 20 μM (reflecting early
   apoptosis) and Bcl-2 downregulation at 40 μM (reflecting late
   apoptosis). As highlighted by Guo et al. (2021) [[166]29],
   morphological changes detected by Hoechst staining occur earlier than
   biochemical markers such as Bcl-2 degradation. Additionally,
   methodological differences in cell density between assays may influence
   drug sensitivity: lower-density cultures used for Hoechst staining are
   more susceptible to apoptosis initiation, whereas higher-density
   cultures required for Western blot analysis may need stronger
   pro-apoptotic stimuli to overcome survival signals. Despite these
   variations, both experiments consistently demonstrate a dose-dependent
   induction of apoptosis by isalpinin, with an alignment of morphological
   and biochemical hallmarks at higher concentrations. This body of
   evidence underscores that PIK3R1 is a key modulator and pharmacological
   target of flavonol-based compounds. Although isorhamnetin and isalpinin
   inhibit the PI3K/Akt pathway through direct or structural binding,
   their methylation profiles enhance both metabolic stability and binding
   selectivity [[167]39]. This provides a compelling rationale for the
   development of methylated flavonols as non-toxic, multi-target
   anticancer agents.

   Based on prior evidence implicating the Akt pathway in the mechanism of
   isalpinin, its direct impact on signaling was assessed in responsive
   NSCLC lines. The selection of H23 and H460 cells for this analysis was
   guided by their differential sensitivities and underlying genetic
   profiles. These cell lines demonstrated a high sensitivity to
   isalpinin, with an IC[50] approximately 2-fold lower than that of A549
   at 48 h. This lower sensitivity of A549 cells is consistent with the
   presence of a KRAS mutation, which is associated with the dominance of
   MAPK signaling and a reduced dependency on the PI3K/Akt pathway
   [[168]40]. Conversely, the high basal p-Akt levels in H460 cells and
   the loss of STK11/LKB1 in H23 cells created a greater reliance on
   PI3K/mTOR pathway signaling [[169]41,[170]42], thereby enhancing their
   sensitivity to inhibition by isalpinin. In this study, isalpinin
   demonstrated in vitro anticancer activity by inhibiting the PI3K/Akt
   pathway through ATP-binding site interaction similar to alpelisib and
   by generating ROS, reflecting established NSCLC therapeutics
   [[171]43,[172]44]. A concentration-dependent suppression of Akt
   (Ser473) and GSK3β (Ser9) phosphorylation was observed, targeting a
   pathway frequently dysregulated in NSCLC [[173]45]. Additionally,
   isalpinin generates reactive oxygen species ROS through a mechanism
   analogous to cisplatin [[174]45,[175]46], achieving potent cytotoxicity
   (IC[50] 44 to 82 μM) while demonstrating superior cancer cell
   selectivity over normal fibroblasts (IC[50] > 100 μM vs. cisplatin
   14.65 μM) in cell-based assays. This preferential activity in H23/H460
   lines correlates with PIK3CA alterations prevalent in 33.1% of lung
   squamous cell carcinomas [[176]42] which sensitize tumors to PI3K/Akt
   pathway modulation [[177]47]. While these in vitro results highlight
   the potential of isalpinin as an anticancer candidate, further in vivo
   validation remains essential to confirm therapeutic efficacy. These
   preliminary findings suggest that isalpinin is a promising candidate
   for drug discovery development and warrants continued investigation as
   a potential therapeutic agent to address the current limitations in
   NSCLC treatment.

   Combination therapeutic strategies are paramount in contemporary
   oncology, aimed at overcoming drug resistance, mitigating adverse
   effects, and improving patient outcomes. Flavonoids exhibit
   considerable promise as both monotherapeutic agents and as adjuvants
   that enhance the efficacy of conventional chemotherapy or
   immunotherapy. For instance, quercetin and kaempferol augment the
   cytotoxic effects of cisplatin by impeding DNA repair pathways (e.g.,
   base excision repair) and inhibiting drug efflux pumps (e.g.,
   P-glycoprotein), thereby increasing intracellular platinum accumulation
   [[178]48,[179]49]. The synergistic potential of flavonols with immune
   checkpoint inhibitors is currently being investigated in clinical
   trials, as exemplified by the ongoing Phase 2 study (ClinicalTrials.gov
   identifier: [180]NCT05724329) evaluating neoadjuvant tislelizumab
   combined with dasatinib and quercetin in resectable head and neck
   squamous cell carcinoma (COIS-01). The distinct mechanisms of action of
   isalpinin, characterized by PI3K/Akt pathway inhibition and reactive
   oxygen species-driven apoptosis, position it as a promising
   combinatorial agent, potentially facilitating reduced cisplatin
   dosages, thereby diminishing nephrotoxicity while preserving efficacy,
   and could act in concert with chemotherapeutic drugs within the tumor
   microenvironment. Integrating flavonoid-driven strategies is crucial
   for addressing critical challenges in precision oncology, particularly
   in tumors with PI3K/Akt dysregulation or immunosuppressive tumor
   microenvironments. Future research needs to be conducted to validate
   the synergy in relevant in vitro models (e.g., H23 and H460: NSCLC
   subtypes) and in vivo-resistant tumor models, and to optimize doses for
   efficacy and safety in flavonoid-chemotherapy and flavonoid-immune
   checkpoint inhibitor pairings.

   This study is the first to demonstrate that isalpinin from P. dianthum
   acts as a multi-target subtype-selective anticancer agent against
   non-small cell lung cancer (NSCLC). This shows that isalpinin inhibits
   cell proliferation, migration, and anchorage-independent growth;
   induces ROS-mediated apoptosis; and suppresses the PI3K/Akt/GSK3β
   signaling pathway, resulting in cell death via apoptosis. These
   findings provide novel mechanistic insights and suggest the potential
   of isalpinin for the preclinical development of lung cancer therapy.

4. Materials and Methods

4.1. Materials

   RPMI-1640, DMEM, L-glutamine, penicillin, streptomycin, fetal bovine
   serum (FBS), 0.25% trypsin-EDTA, and phosphate-buffered saline (PBS)
   were purchased from GIBCO (Gaithersburg, MA, USA). A quantity of
   3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was
   purchased from Invitrogen (Carlsbad, CA, USA). Dimethyl sulfoxide
   (DMSO), Hoechst 33342, 2′-7′- dichlorodihydrofluorescein diacetate
   (DCFH[2]-DA) were obtained from Sigma (St. Louis, MO, USA). Agar nobles
   were purchased from BD Biosciences (Franklin Lakes, NJ, USA). Primary
   antibodies were used as follows: Rabbit anti-phosphorylated Akt
   (Ser473; CST, Beverly, MA, USA); rabbit anti-Akt (CST, Beverly, MA,
   USA); rabbit anti-phosphorylated GSK-3β (Ser9; CST, Beverly, MA, USA);
   rabbit anti-GSK-3β (CST, Beverly, MA, USA); rabbit anti-GAPDH (CST,
   Beverly, MA, USA) and rabbit anti-Bcl-2 (CST, Beverly, MA, USA). The
   secondary antibody used in this study was anti-rabbit IgG HRP-linked
   (CST, Beverly, MA, USA).

4.2. Methods

4.2.1. Isalpinin Preparation

   Isalpinin ([181]Figure 1, [182]Supplementary S1) was isolated from the
   roots and leaves of P. dianthum as previously described [[183]13]. For
   isalpinin preparation, it was dissolved in DMSO as a stock solution,
   which was further diluted with cell culture medium to the desired
   working concentrations, and the control samples were incubated with
   0.1% DMSO in the culture medium. The final concentration of DMSO was
   less than 0.1%, indicating no toxicity. Control cells exposed to equal
   concentrations of DMSO were used for comparison with the effect of the
   isalpinin-treated group.

4.2.2. Cell Culture

   Human non-small cell lung cancer H460 (HTB-177), H23 (CRL-5800), A549
   (CCL-185), as well as the normal mouse embryonic fibroblast cell line
   NIH/3T3 (CRL-1658) were obtained from American Type Culture Collection
   (ATCC) (Manassas, VA, USA). H460 and H23 cells were cultured in
   RPMI-1640 medium and A549 cells were cultured in DMEM (Gibco,
   Gaithersburg, MA, USA). All cell cultures were supplemented with 10%
   fetal bovine serum (FBS), 2 mM L-glutamine, and 100 units/mL
   penicillin/streptomycin and incubated at 37 °C in a humidified
   atmosphere of 5% CO[2] and 95% humidity stream up to 70–80% confluence
   before use in the experiments.

4.2.3. Cytotoxicity and Cell Proliferation Assays

   The cytotoxic effects of isalpinin were assessed using the MTT
   colorimetric assay, following the method previously described by
   [[184]50]. Briefly, cells were seeded in 96-well plates at a density of
   1 × 10^4 cells per well and allowed to adhere overnight at 37 °C in a
   humidified incubator with 5% CO[2]. The following day, the cells were
   treated with increasing concentrations of isalpinin (0–200 μM) or
   cisplatin (0–250 μM), as a positive control for 24 and 48 h. After the
   treatment period, the cells were washed with phosphate-buffered saline
   (PBS), and 100 μL of MTT solution (0.5 mg/mL) was added to each well.
   The plates were incubated for an additional 3 h to allow formazan
   crystal formation. Subsequently, 200 μL of DMSO was added to each well
   to solubilize the crystals, and absorbance was measured at 570 nm using
   a microplate reader (Perkin Elmer VICTOR3/Wallac1420). Cell viability
   was calculated as a percentage of the untreated control, and the IC[50]
   values (concentration required to inhibit 50% of cell viability) were
   determined using nonlinear regression analysis in GraphPad Prism
   version 8 (GraphPad Software, CA, USA).

   To assess cell proliferation, a low seeding density of 2 × 10^3 cells
   per well was used in 96-well plates. Cells were allowed to adhere
   overnight and were subsequently treated with a non-cytotoxic
   concentration of isalpinin for 48 h, encompassing approximately two
   cell doubling times. Cell growth was measured using the MTT assay
   protocol. The results were expressed as the percentage of cell
   proliferation in the treated wells relative to that in the control
   wells.

4.2.4. Cell Migration Assays

   Cell migratory capacity was evaluated using a wound-healing assay, as
   previously described with modifications [[185]38,[186]51]. The assay
   was employed to assess the inhibitory effect of isalpinin on the
   migration of non-small cell lung cancer cell lines H23, H460, and A549.
   Briefly, cells were seeded into 24-well plates at a density of 2 × 10^5
   cells per well and allowed to adhere overnight in a complete culture
   medium. Upon reaching confluence, a uniform linear scratch was
   introduced across the monolayer using a sterile 200 μL pipette tip to
   simulate a wound. The detached cells and debris were carefully removed
   by washing with PBS buffer 500 μL. Subsequently, the cells were treated
   with varying concentrations of isalpinin (0–40 μM) and incubated for
   several hours. Wound closure was monitored using phase-contrast
   microscopy with a Nikon Ts2 (Tokyo, Japan) digital microscope. Images
   were captured 0, 24, and 48 h post-scratch. The wound area was
   quantified using ImageJ software version 1.53 (National Institutes of
   Health, Bethesda, USA), and the relative wound area was recalculated.
   The rate of cell migration was determined by calculating the value from
   the regression analysis of wound closure area the 48 h period. All
   experiments were conducted in triplicate to ensure reproducibility.

4.2.5. Soft Agar Colony Formation Assay

   The ability of isalpinin to inhibit anchorage-independent growth and
   survival of non-small cell lung cancer cell lines (A549, H23, and H460)
   was evaluated using a soft agar colony formation assay, as previously,
   described with modifications [[187]52]. A two-layer soft agar system
   was established in 24-ultra-low-attachmenthment plates (Corning, NY,
   USA). The bottom layer consisted of 250 μL of 1.2% Noble agar
   (Sigma-Aldrich, Louis, MO, USA) prepared in DMEM or RPMI-1640 medium
   supplemented with 5% FBS. This layer was allowed to dry at 37 °C for 2
   h. The top layer was composed of 0.6% agar containing 1.5 × 10^3 cells
   per well (A549, H23, or H460) in the same medium, supplemented with
   either a non-cytotoxic concentration of isalpinin or 0.1% (v/v) DMSO as
   a vehicle control. Following solidification of the top agar layer, 250
   μL of complete medium was added to each well to maintain hydration and
   nutrient availability. The cultures were incubated at 37 °C in a
   humidified incubator with 5% CO[2] for 14 days. The culture medium was
   carefully replenished every 3 days. At the end of the incubation
   period, the colonies were visualized using a light microscope equipped
   with a digital camera. Images were captured and analyzed to quantify
   both colony number and size, which reflect the extent of
   anchorage-independent cell proliferation. A quantitative analysis was
   performed using Axiovision 4 software (Carl Zeiss MicroImaging GmbH,
   Jena, Germany) and I (Institute of Health, Bethesda, MD, USA).

4.2.6. Determination of Reactive Oxygen Species (ROS)

   Intracellular reactive oxygen species (ROS) levels were quantified
   using the DCFH[2]-DA assay [[188]37]. Briefly, cells (7.5 × 10^4
   cells/well) were seeded as a monolayer in 24-well plates and incubated
   overnight. After washing with PBS, the cells were incubated with 100 μM
   of DCFH[2]-DA solution for 30 min. at 37 °C. Subsequently, the cells
   were treated with isalpinin (0–40 μM) or 600 μM hydrogen peroxide
   (H[2]O[2]) as a positive control for 1 h. After treatment, the cells
   were lysed with 1% Triton X-100 and the fluorescence intensity of
   oxidized DCF was measured at 485 nm excitation and 535 nm emission
   using a microplate reader (Perkin Elmer VICTOR3/Wallac1420).

4.2.7. Cell Apoptosis

   Apoptotic cell death was evaluated using Hoechst 33342, a fluorescent
   nuclear staining dye [[189]36]. A549, H23, and H460 cells were seeded
   at a density of 8 × 10^3 cells/well in 96-well plates and allowed to
   adhere overnight. The cells were treated with isalpinin for 48 h.
   Following treatment, cells were incubated with Hoechst 33342 (10 μg/mL)
   for 30 min in the dark at room temperature. Nuclear morphological
   changes characteristic of apoptosis, including chromatin condensation
   and DNA fragmentation, were visualized using a fluorescence microscope
   (BX-FLA; Olympus, Tokyo, Japan) at 20× magnification with
   excitation/emission wavelengths of 350/461 nm. Apoptotic cells were
   quantified and expressed as a percentage of the total cell population.
   All experiments were conducted in triplicate to ensure reproducibility.

4.2.8. Western Blot Analysis

   A total of 8 × 10^5 cells were seeded into 60-mm culture dishes and
   incubated overnight to allow attachment. The cells were treated with
   various concentrations of isalpinin for 24 h. Following treatment,
   cells were lysed on ice for 30 min using LIPA buffer supplemented with
   a protease inhibitor cocktail (Corning, NY, USA). Lysates were
   clarified by centrifugation at 12,000× g for 15 min at 4 °C and the
   resulting supernatants were collected. The Total protein concentration
   was determined using a BSA protein assay kit (Thermo Fisher Scientific,
   Waltham, MA, USA). The protein samples were denatured by boiling in 2X
   sample buffer at 95 °C for 5 min. Equal amounts of protein (40 μg per
   lane) were separated using SDS-polyacrylamide gel electrophoresis
   (SDS-PAGE) and subsequently transferred onto polyvinylidene fluoride
   (PVDF) membranes. Membranes were blocked in 5% skim milk prepared in
   Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h at room
   temperature, followed by overnight incubation at 4 °C with rabbit
   primary antibodies. After washing, the membranes were incubated with
   appropriate HRP-conjugated secondary antibodies at room temperature for
   2 h. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the
   loading control. Protein bands were visualized using enhanced
   chemiluminescence (ECL) with Immobilon Western Chemiluminescent HRP
   Substrate (Millipore, MA, USA) and quantified using ImageJ software
   (NIH, Bethesda, MD, USA).

4.2.9. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
Analysis

   Transcriptomic data from NSCLC cell lines were analyzed alongside
   public NSCLC datasets (TCGA) to identify dysregulated pathways. GO
   [[190]53] and KEGG [[191]54] analyses were performed using the STRING
   11.5 database [[192]55], with relationships between gene count and
   adjusted p-value visualized via scatterplots. R software version 4.2.3
   with ggplot2 [[193]56] was used to generate bubble scatterplots of
   enriched GO terms and KEGG pathways [[194]57]. Potential targets of
   isalpinin were predicted using molecular docking against proteins in
   the PI3K/Akt pathway, validated against structural data from PubChem
   and activity profiles in ChEMBL. Functional annotation and statistical
   analyses employed standard bioinformatics tools, with p-values < 0.05
   as the significance threshold. These integrated analyses provided
   mechanistic insights into the multitarget effects of isalpinin,
   highlighting its regulation of key biological processes (e.g.,
   apoptosis, proliferation) and signaling pathways (e.g., PI3K/Akt)
   relevant to NSCLC.

4.2.10. Construction of the Protein–Protein Interaction (PPI) Network and
Computational Modeling of AKT with Molecular Docking

   To investigate the protein interaction landscape, a protein–protein
   interaction (PPI) network was constructed using the STRING database
   (version 11.0; [[195]55] [196]https://string-db.org/) [[197]58]. Gene
   targets of interest were input under the Homo sapiens organism model
   and a high-confidence interaction score threshold of 0.950 was applied.
   In the resulting network, nodes represented proteins and edges denoted
   by protein–protein associations [[198]59]. To identify core target
   proteins, topological parameters, including degree centrality,
   betweenness centrality, closeness centrality, and clustering
   coefficient, were calculated and analyzed using Cytoscape software.

   For molecular docking analysis, the X-ray crystallographic structure of
   activated Akt was retrieved from the Protein Data Bank (PDB ID: 1O6L).
   The chemical structure of isalpinin was generated using ChemDraw Ultra
   17.0 (PerkinElmer, Waltham, MA, USA) and the canonical SMILES format
   was obtained from the ChEBI database [[199]60]. The potential binding
   interactions between isalpinin and AKT were evaluated using molecular
   docking simulations. To determine the docking site, CurPocket, a
   curvature-based cavity detection algorithm, was used to identify the
   potential binding pockets on the AKT protein surface. Molecular docking
   was subsequently performed using AutoDock Vina (version 1.1.2)
   integrated into the CB-Dock2 platform, which enables blind docking
   simulations [[200]61]. The binding poses with the most negative Vina
   score, which is indicative of the highest binding affinity, was
   selected for further analysis. Detailed binding parameters, including
   the docking center, grid size, interacting residues, and molecular
   contacts, were examined. The resulting protein–ligand interactions and
   docking conformations were visualized using BIOVIA Discovery Studio
   Visualizer (BIOVIA, San Diego, CA, USA), providing structural insight
   into the binding mechanism of isalpinin with AKT.

4.2.11. Statistical Analysis

   Data are presented as mean ± standard error of the mean (SEM) from at
   least four independent experiments, and all data were analyzed using
   Prism 8 (GraphPad Software, Inc., San Diego, CA, USA). One-way analysis
   of variance (ANOVA) with Tukey’s Multiple Comparison Test was used to
   determine statistical significance between the control and treatment
   groups, with p-values < 0.05.

5. Conclusions

   In summary, this study demonstrates that isalpinin, a flavonol derived
   from P. dianthum, exhibits potent anticancer activity against non-small
   cell lung cancer (NSCLC) cell lines. At concentrations of 40 μM,
   isalpinin significantly inhibited cell viability, proliferation, and
   migration, while promoting apoptosis particularly in H23 and H460
   cells. Mechanistically, isalpinin induced apoptosis via ROS generation,
   Bcl-2 downregulation, and the suppression of the Akt/GSK3β signaling
   pathway. These findings align with growing evidence supporting the role
   of natural flavonoids in modulating key oncogenic pathways in lung
   cancer. The marked sensitivity of H23 and H460 cells underscores the
   potential of isalpinin as a subtype-specific therapeutic candidate for
   NSCLC. Further in vivo validation and combination studies with standard
   therapies are warranted to advance the clinical development of
   isalpinin as a novel anticancer agent.

Acknowledgments