Alizarin induces a multidirectional mechanism of anti-cancer action in cervical cancer and prostate cancer cells.

Introduction
Anthraquinones are the largest group of naturally occurring quinones many of which occur in plants of the Rubiaceae family used in traditional Chinese medicine for their anti-inflammatory, antioxidant, immunomodulatory, neuroprotective, and antibacterial effects1. Examples include Radix rubiae and Rubia cordifolia L., and anthraquinones isolated from their roots and rhizomes have been widely used in China for thousands of years, among others, in the treatment of joint pain and various types of bleeding2. Phytochemical studies have shown that more than a hundred chemically active ingredients have been isolated from the above-mentioned plants, including anthraquinones such as alizarin, purpuroxanthin, purpurin, ruberythric acid, naphthoquinones, triterpenoids, and cyclic hexapeptides3.
Currently, anthraquinones of both natural and synthetic origin are components of many drugs with a wide range of applications. One of the anthraquinones with multidirectional action is alizarin (2-dihydroxyanthraquinone) which has antimicrobial activity demonstrated by inhibiting biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis strains4. Alizarin also inhibits the genotoxicity of mutagens such as heterocyclic amines and PAHs, which is related to its ability to inhibit CYP activity (CYP1A1, CYP1A2 and CYP1B1) responsible for the activation of mutagens5. It can also be used as an osteotropic drug in the treatment of bone cancer due to its strong antiproliferative effect on osteosarcoma cells, inhibition of ERK phosphorylation and arrest of the cell cycle in the S phase. It also has an affinity for calcium, thus achieving high target specificity for bone tissue1. Furthermore, alizarin and other anthraquinones (purpurine, quinalisarin, and danthrone) have demonstrated potential as GST enzyme inhibitors in this tumor6. Its potential anticancer activity was also observed in colon cancer cells7. Alizarin may also play an important role in reversing gemcitabine resistance caused by excessive activation of NF-κB in pancreatic cancer cells2. Its derivative alizarin 2-O-methoxide also demonstrated antiproliferative activity in gastric, cervical and liver cancer cells8.
Currently, there is a lack of data on the effect of alizarin on cervical and prostate cancer cells, which was the focus of our study. The selection of cell lines was based on epidemiological data indicating that cervical and prostate cancers are among the most common malignancies associated with high mortality worldwide. The selected lines are typically used to test the efficacy of new potential anticancer drugs and therapies, so in our studies, we also assessed the activity of alizarin conjugated with a Bcl-2 inhibitor, Venetoclax, to demonstrate their synergistic effects. This seems appropriate, especially since recent reports suggest potential use of Venetoclax in the treatment of cervical and prostate cancer, including in combination therapies9,10. Recent studies suggesting the possibility of combining Venetoclax with phytochemicals, including curcumin and quercetin11, also seem interesting.

Results

Alizarin has cytotoxic effects on HeLa and DU145 cells
The MTT test showed significant (p ≤ 0.0001) inhibition of cell viability, which was demonstrated by the inhibition of their ability to reduce MTT dye (Fig. 1A). At a concentration of 150 µM, the viability of HeLa cells was 61.2% and 65.29% (DU145 line). Higher concentrations of 250 and 300 µM caused a further reduction in viability to 52.16% and 26.93% (HeLa line) and to 57.81% and 34.31% (DU145 line), respectively. The lowest cell viability was obtained at 350 µM, i.e. 12.79% for the HeLa line and 13.83% for the DU145 line. The control was assumed as 100%.
Alizarin cytotoxicity was also demonstrated in FDA/PI staining, where at 350 µM a significant increase in the number of dead cells was demonstrated (88%—HeLa line, 86%—DU145 line). Dead cells (labeled with propidium iodide) had a red fluorescent color due to binding to DNA after membrane damage. Control cells (live) were characterized by green fluorescence emission from fluorescein diacetate (Fig. 1F,G).

Pro-apoptotic effect of alizarin in HeLa and DU145 cell lines
Exposure of the studied cells to alizarin caused an increase in the number of early- (annexin V-PE + /7-AAD) and late-apoptotic cells (annexin V-PE + /7-AAD +). In the case of the HeLa line, at a concentration of 150 µM, apoptotic cells constituted 43.5% (p ≤ 0.0001), and at 250 uM over 50% (p ≤ 0.0001) (Fig. 1B,C). The highest percentage of apoptotic cells (late apoptotic phenotype) was noted at concentrations of 300 µM and 350 µM up to 71.45% and 89.15% (p ≤ 0.0001). The consequence of exposure of DU145 cells to alizarin (150 µM) was a significant (p ≤ 0.0001) increase in the number of apoptotic cells (41.35%), which progressed with the increase in the concentration of the tested compound to 47.6% at 250 µM and to 66.95% (300 µM) (Fig. 1B′,C). At 350 µM, over 88.1% (p ≤ 0.0001) of the pool of all cells were apoptotic cells. The activation of caspases 3/7 confirmed the apoptosis process. The highest activity of executive caspases was demonstrated at a concentration of 350 µM, i.e. 85.9%, p ≤ 0.0001 (HeLa line, Fig. 1D,E) and 83.9%, p ≤ 0.0001 (DU145 line, Fig. 1D′,E) of caspase-positive cells. The results indicate a proapoptotic effect of alizarin including activation of executive caspases.

Alizarin shows cytotoxicity in spheroids (3D model)
After 4 days of spheroid growth, their diameter was measured, which was on average 759.24 µm for HeLa line cells and 761.93 for DU145 line cells. In the spheroids from the control group, in accordance with the morphological structure, an outer layer was shown, which consisted of live cells (green emission-FDA) and an inner layer of dead cells (red emission-PI) (Fig. 1F′). On the other hand, spheroids after the action of alizarin (350 µM/48h) were characterized by an increased number of dead cells, visible disintegration of the spheroid structure and the presence of numerous small cell aggregates. Cytometric analysis showed that apoptotic cells constituted 62.1% for the HeLa line and 63.4% for the DU145 line (Fig. 1H,I). This proves the toxicity of alizarin in relation to the spheroids formed in the 3D model.

The effect of alizarin on VH10 cell line
Additionally, alizarin cytotoxicity was analyzed on normal fibroblasts-the VH10 cell line (Supplementary Figure S1). The MTT assay demonstrated that alizarin exhibited low cytotoxicity to the tested cells. At a concentration of 350 µM, 30% of dead cells were observed. The obtained results correlated with the observed level of apoptosis (Annexin V test) where a concentration-dependent increase in the number of apoptotic cells was observed (at 350 µM, 32% of apoptotic cells were shown). Hence the remaining studies were carried out on HeLa and DU145 cell lines.

Alizarin induces an increase in the level of Bcl-2 phosphorylation
Alizarin treatment resulted in a concentration-dependent increase in the phosphorylation level of Bcl-2 at Ser70 in HeLa and DU145 cell lines (Fig. 2). It has been reported that phosphorylation of Bcl-2 at Ser70 typically enhances its antiapoptotic activity, particularly in response to stress factors12. Therefore, the observed increase in Bcl-2 phosphorylation is consistent with attenuation of its antiapoptotic activity. At concentrations of 100 and 200 µM, the relative level of phosphorylated Bcl-2 increased to 42.15% and 52.89%, respectively (p ≤ 0.0001) in HeLa cells (Fig. 2A,C), and to 45.96% and 58.56%, respectively (p ≤ 0.0001) in DU145 cells (Fig. 2A′,C). These results indicate that alizarin treatment is associated with enhanced phosphorylation of Bcl-2 at Ser70, which may contribute to increased apoptotic susceptibility of cancer cells.

Alizarin induces phosphorylation of H2A.X and ATM
48-h incubation of cells with alizarin led to increased phosphorylation of H2A.X and ATM (Fig. 2), which is a marker of the cellular response to DNA double-strand breaks (DSBs). However, it should be noted that the observed H2A.X/ATM phosphorylation may not necessarily reflect direct DNA damage, but may also result from secondary activation of caspase 3 during apoptosis. In HeLa cells, at concentrations of 150 and 250 µM, the percentage of cells with phosphorylated H2A.X was 35.88% and 46.05% (p ≤ 0.0001) (Fig. 2B,D), respectively, and in prostate cells 15.25% and 56.25% (p ≤ 0.0001) (Fig. 2B′,D). The highest percentage of cells with H2A.X and ATM activation was observed at the concentration of 350 µM (HeLa: 93.47%, DU145: 80.69%), which correlated with the level of apoptosis.

Alizarin has a pro-oxidant effect
The assessment of the level of oxidative stress in the tested cells exposed to alizarin was based on intracellular detection of superoxide radicals (Fig. 3). A significant (p ≤ 0.0001) increase in ROS production was demonstrated, dependent on the alizarin concentration. At a concentration of 350 µM, the highest percentage of ROS (+) cells was noted, i.e. over 68% for HeLa cells (Fig. 3C,D) and 64% for DU145 (Fig. 3C′,D). The obtained results indicate the induction of oxidative stress by the tested anthraquinone.
Increased production of reactive oxygen species correlated with decreased mitochondrial membrane potential (Fig. 3). In HeLa cells, the percentage of cells with mitochondrial membrane depolarization at a concentration of 150 µM was 21.75% (p ≤ 0.0001) (Fig. 3A,B). At concentrations of 250 µM and 300 µM, 48.4% and 70.8% (p ≤ 0.0001) of cells showed decreased mitochondrial membrane potential, respectively. Alizarin exposure at a concentration of 350 µM resulted in the highest percentage of cells with depolarization exceeding 80% (p ≤ 0.0001). Similar changes were observed in DU145 cells (Fig. 3A′,B). The percentage of cells with reduced mitochondrial potential, depending on the concentration of the tested compound, was: 18.94% (150 µM), 47.35% (250 µM), 69.55% (300 µM) and 76.45% at 350 µM.

Alizarin induces changes in the ultrastructure of the studied cells
Alizarin in HeLa cells (150 µM) caused mitochondrial swelling, growth and swelling of the Golgi apparatus, and an increase in the number of autophagic vacuoles (Fig. 4A). Alizarin at a concentration of 250 µM influenced further changes in mitochondria, including swelling and reduction of mitochondrial cristae. Swelling of the channels of the rough endoplasmic reticulum and an increased number of autophagic vacuoles and autophagolysosomes were also observed. At 300 µM, increased autophagic changes were observed, as indicated by numerous primary lysosomes, autophagic vacuoles, autophagolysosomes, and secondary lysosomes. Mitochondria were characterized by significant swelling. At a concentration of 350 µM, mitochondria were shown to be characterized by structural disorganization and significant damage. Damaged mitochondrial cristae (black arrow) were observed in the electron-lucent mitochondrial matrix (Fig. 4A). Significant swelling of the reticulum channels was also shown, indicating its stress.
Ultrastructural changes correlated with morphometric measurements. A progressive increase in mitochondrial size was shown with increasing alizarin concentration. At a concentration of 150 µM, mean mitochondrial length was 0.74 µm and 0.85 µm at a concentration of 250 µM compared to the control (0.57 µm). The greatest mitochondrial swelling was shown at a concentration of 300 and 350 µM, to 1.12 µm and to 1.28 µm, respectively (Fig. 4B).
A characteristic feature of the ultrastructure of DU145 cells was the intensification of autophagic processes (Fig. 4A′). At a concentration of 150 µM, an increased number of autophagic vacuoles at various stages of digestion and swollen mitochondria were observed. Changes in the structure of the cell nucleus were also demonstrated, concerning changes in its shape and progressive swelling and damage to mitochondria (250 µM). Vacuole-like structures were also visible in the mitochondrial matrix, which are a direct result of osmotic swelling of mitochondria in response to cellular stress. This process is closely linked to oxidative stress-induced mitochondrial dysfunction. As a result of exposing cells to alizarin at concentrations of 300 and 350 µM, an increased number of autophagic vacuoles and secondary lysosomes, including lamellar structures corresponding to late autophagolysosomes, which function as the final degradation bodies in autophagy, was observed. Various stages of vacuolar degeneration of mitochondria were also observed, occurring in the form of concentric reconstruction of mitochondrial cristae (Fig. 4A′). The channels of the rough reticulum were characterized by significant swelling. They appeared as long, swollen, and electron-lucent channels. Mean mitochondrial lenght measurements performed for concentrations of 150 and 250 µM were 0.8 and 0.95 µm, respectively. Similarly to the HeLa line, the greatest mitochondrial swelling was demonstrated at concentrations of 300 and 350 µM, i.e. 1.1 µm and 1.2 µm (Fig. 4B).

Alizarin modulates the assessment of LC3-II protein level
The presence of numerous autophagosomes in the examined cells, demonstrated by electron microscopy, was confirmed by analysis of the LC3-II protein level (Supplementary Fig. S2). At 150 µM, the mean fluorescence intensity of LC3-II–associated Alexa Fluor® 555 signal in HeLa cells increased to 126.16%, while at DU145 to 135.44% relative to control. As a result of using a concentration of 250 µM, a further increase in intensity was observed to 139.88% (HeLa line) and to over 153% (DU145 line). A gradual reduction in the emission of the dye in the labeled cells was demonstrated at concentrations of 300 µM (90.34%-HeLa and 89.53%-DU145) and 350 uM to 85.26% (HeLa) and 79.68% for the DU145 line cells. The obtained results of flow cytometric analysis indicate the degradation of LC3-II by lysosomal enzymes, which confirms alizarin-induced macroautophagy.

Alizarin increases vacuolization and apoptotic changes-morphological assessment
Morphological analysis revealed alizarin concentration-dependent cytoplasmic vacuolation and induction of apoptosis (Fig. 5A,A′). At 150 µM, a significant increase in the number of cells with vacuoles was demonstrated (1399.36 for the HeLa line and 1405.33 cells for the DU145 line, p ≤ 0.0001). At concentrations of 250–350 µM, there was a progressive decrease in the number of cells with cytoplasmic vacuolization to 16.37% in favor of an increase in the number of apoptotic cells, which constituted over 80% of the pool of all analyzed cells (350 µM/48h) (Fig. 5B,C). Analogous changes were observed in the case of the DU145 line. In this case, there was also a significant decrease in the number of cells with cytoplasmic vacuolization and an increase in the number of apoptotic cells. At a concentration of 350 µM, apoptotic cells constituted about 80%, and with vacuolation over 13% (Fig. 5B,C). The observed apoptotic cells were characterized by typical morphological changes such as chromatin condensation and fragmentation, shrinkage and thickened, darkly stained cytoplasm. The demonstrated number of apoptotic cells correlated with the cytometric analysis (annexin V test).

Alizarin induces mitotic death
Alizarin showed a statistically significant increase in the indicators of mitotic catastrophe, i.e. the number of binucleated cells, multinucleated cells, giant cells and cells with micronuclei (Fig. 5A,A′,D). Giant cells were characterized by large size, increased number of cell nuclei. Often, numerous micronuclei and cytoplasmic vacuoles were also present in the cytoplasm. The presence of abnormal mitotic figures was also characteristic of mitotic death. Mitotic catastrophe marker cells were also observed at the stage of apoptotic death, which is its final stage13. The increase in the above indicators indicates changes in the mitotic apparatus as a consequence of the action of alizarin.

Alizarin blocks cells in the G2/M phase and reduces the mitotic index
Flow cytometric analysis showed that alizarin caused a progressive blockade of cells in the G2/M phase of the cell cycle with increasing concentration. The greatest increase in the cell population in the G2/M phase was observed at a concentration of 350 µM, i.e., 56.5% for the HeLa line (34.1% of cells in the control group) and 53.9% for the DU145 line (29.1% of cells in the control group), with p ≤ 0.0001 (Fig. 6A,B).
The use of increasing concentrations of alizarin resulted in a statistically significant reduction in the mitotic index (Fig. 6C). At concentrations of 150 µM and 250 µM, the mitotic index was 43% and 26.7% for the HeLa line and 57.05% and 46.54% for DU145, respectively, with p ≤ 0.0001. The highest concentration of alizarin (350 µM) caused the greatest reduction in the number of dividing cells, i.e. MI = 6.63% (HeLa line) and 15.67% (DU145) (p ≤ 0.0001). The control value was taken as 100%.

Alizarin affects the migration of HeLa and DU145 cell lines
Changes in the ability of cells to migrate induced by alizarin were observed at two time points, i.e. 24 and 48 h after the formation of a “torn wound” constituting a cell-free space to which cells can migrate, which mimics the wound healing process. In the control group (HeLa line), a more than 50% reduction in wound width was demonstrated after 24 h (148.84 µm) and further "healing of the monolayer" of cells after 48 h to approx. 75% (84.67 µm) (Fig. 6D,E). At point 0, the width of the resulting scratch was 330 µm. 48-h incubation of cells with alizarin at concentrations of 150 and 250 µM resulted in 46.38% (183.89 µm) and 47.71% (184.56 µm) of monolayer overgrowth. The lowest percentage of cell migration was shown at a concentration of 350 µM to 8% (316.85%) after 48 h.
Alizarin also affected the inhibition of DU145 cell migration (Fig. 6D′,E). In the control group, cells covered more than 66% (111 µm) to 87% (43 µm) of the wound surface after 24 and 48 h, respectively. The width of the “torn wound” at point 0 was 332.73 µm. The surface overgrowth was found to be 45.39% (184.56 µm/24 h) and 54% (155.47 µm/48 h) at the concentration of 150 µM, while for the concentration of 250 µM it was 22.26% (244.09 µm/24 h) and 47.07% (177.96 µm/48 h). Similarly to the HeLa line, the highest inhibition of cell migration was demonstrated at 350 µM, where the scratch overgrowth was 24.41% (254.04 µm/24 h) and 33.93% (222 µm/48 h). The obtained results indicate the inhibitory effect of alizarin on the migration of HeLa and DU145 cell lines.

Alizarin inhibits the clonogenic capacity of HeLa and DU145 cells
A colony formation test was used in the studies to assess the ability of adherent cells to proliferate and form colonies. The results showed a significant reduction in the number of cell colonies in the groups treated with alizarin, and this effect depended on its concentration (Fig. 6F,F′). The concentration of 150 µM clearly inhibited the formation of cell colonies in both the HeLa (45.85%, Fig. 6E,F) and DU145 (75.25%, Fig. 6E,F′) lines. A further reduction in colony formation occurred with an increase in the concentration of the tested anthraquinone to 9.89% (250 µM) and to 6.33% (300 µM) for the HeLa line, as well as to 17.11% (250 µM) and to 9.33% (350 µM) for the DU145 line. At a concentration of 350 µM, cell colony formation was practically inhibited (3.49%-Hela and 4.08%-DU145). Alizarin has a strong antiproliferative effect on the tested cells.

Alizarin and Venetoclax act synergistically to reduce viability and enhance apoptosis
Our results showed that 48-h treatment with alizarin (250 µM) in combination with Venetoclax increased the inhibition of the viability of the tested cells to 39.29%-HeLa and 33.65%-DU145, p ≤ 0.0001 (ALIZ 250 µM + VENET 4 µM) and to 27.34%-HeLa and 19.73%-DU145, p ≤ 0.0001 (ALIZ 250 µM + VENET 8 µM) (Fig. 7A). For Venetoclax at a concentration of 4 and 8 µM, cell viability was at the level of 75 and 63.2% (HeLa line) and 66.89 and 61.4% (DU145 line).
Their combined action (ALIZ 250 µM + VENET 8 µM) also increased the level of apoptosis to 71.45% (HeLa line, Fig. 7B,C) and 79.98% (DU145 line, Fig. 7B′,C) and increased the number of cells with active executive caspase 3/7 to 74.78% (HeLa line, Fig. 7D,E) and 76.01% (DU145 line, Fig. 7D′,E), while for alizarin alone at a concentration of 250 µM the level of apoptosis was about 50%.
Analogous results were observed when analyzing the phosphorylation status of the Bcl-2 protein. Venetoclax at concentrations of 4 and 8 µM resulted in a decrease in phosphorylated Bcl-2 levels by 29.6% and 33.1% in HeLa cells (Fig. 7F,G) and by 28.8% and 30.2% in DU145 cells (Fig. 7F′,G), respectively. Forty-eight-hour treatment with alizarin in combination with Venetoclax further reduced the level of phosphorylated Bcl-2 in both cell lines. Combined treatment with ALIZ 250 µM + VENET 4 µM decreased phosphorylated Bcl-2 by 55.3% in HeLa cells and 59.6% in DU145 cells. At the higher combination (ALIZ 250 µM + VENET 8 µM), phosphorylated Bcl-2 levels dropped by 79.6% in HeLa cells and 80.3% in DU145 cells (p ≤ 0.0001). These results indicate that alizarin and Venetoclax promote proapoptotic signaling, at least in part, by reducing Bcl-2 phosphorylation.

Alizarin and Venetoclax modulate the dual PI3K/MAPK signaling pathway in DU145 and HeLa cell lines
The obtained results showed that cells exposed to alizarin and its combined action with Venetoclax enhanced the proapoptotic effect in the studied cell lines. Therefore, a more comprehensive understanding of their mechanism of action was necessary by analyzing the PI3K/MAPK signaling pathway.
In HeLa and DU145 cells, the effect of alizarin at a concentration of 250 µM was associated with a reduced proportion of cells exhibiting dual Akt and ERK phosphorylation (pAkt⁺/pERK⁺), accompanied by an approximately 50% increase in the double-negative population (pAkt⁻/pERK⁻) (p ≤ 0.0001) (Supplementary Figure S3). These changes coincided with increased apoptotic responses observed in both cell lines. In the case of Venetoclax at a concentration of 4 µM, a moderate effect of dual phosphorylation of Akt and ERK was observed, as well as a transient, compensatory increase in ERK activation (by 11.1% in HeLa cells and slightly higher in DU145 cells (by 13.2%), which decreased after the application of the higher concentration (8 µM). Alizarin conjugated with Venetoclax significantly inhibited the activity of both pathways, as expressed by an 80% (p ≤ 0.0001) increase in the number of double-negative cells (pAkt⁻/pERK⁻) and correlated with the obtained percentage of cells with Bcl-2 phosphorylation.
The obtained results showed that the combined treatment resulted in significantly stronger effects compared to alizarin alone. Combined exposure to alizarin and Venetoclax was associated with alterations in PI3K/Akt and MAPK/ERK signaling profiles in both cell lines, with the extent of pathway modulation correlating with the degree of Bcl-2 phosphorylation.
In summary, both HeLa and DU145 cells showed increased dual-pathway inactivation (pERK⁻/pAkt⁻) following combined treatment with alizarin and Venetoclax compared to single treatment. The effect of the combination was dose-dependent (greater at 8 µM) and correlated with the observed Bcl-2 phosphorylation.

Alizarin and Venetoclax interaction analysis (Bliss independence model)
The interaction between alizarin and Venetoclax was assessed using Bliss’s analysis of independence. In HeLa cells, the combination of alizarin 250 µM with Venetoclax 4 µM induced an additive effect (Fa_observed = 0.6071), whereas the combination with Venetoclax 8 µM showed a slight synergistic tendency (Fa_observed = 0.7266). In DU145 cells, both combinations exceeded the expected effect, indicating a moderate (ALIZ 250 µM + VENET 4 µM) (Fa_observed = 0.6635) to strong (ALIZ 250 µM + VENET 8 µM) (Fa_observed = 0.8027; Fa_expected = 0.668) synergistic interaction. These results indicate that alizarin potentiates the cytotoxic effect of Venetoclax, particularly in prostate cancer cells, likely by promoting sensitivity to apoptosis.

Alizarin and Venetoclax exhibit cytotoxicity in spheroids (3D model)
After 48 h of incubation of HeLa and DU145 spheroids treated with alizarin (250 µM) and Venetoclax (4 and 8 µM) (Fig. 8C,C′), an increased number of dead cells was observed (red emission from PI). Changes in their structure were also observed, including loss of compactness and integrity. Their combined action (with increasing Venetoclax concentration) resulted in a visible disintegration of the spheroid structure, including the presence of smaller cell aggregates. These changes were observed in spheroids formed from both HeLa and DU145 cells. Furthermore, flow cytometric analysis showed that alizarin and Venetoclax (8 µM), similarly to cells in 2D culture, increased apoptosis by more than 30%, although these values were slightly lower. However, the combined action of alizarin (250 µM) and Venetoclax, especially at a concentration of 8 µM, resulted in a statistically significant increase in the number of apoptotic cells to over 60% in both HeLa (Fig. 8A,B) and DU145 µM (Fig. 8A′,B) cell lines. The combined action of alizarin and Venetoclax increased the toxicity in the tested spheroids.

Discussion
Despite the rapidly developing therapies of oncological treatment, i.e. radiotherapy, drug therapy, surgery and local therapy improving the quality of life of cancer patients, certain threats still exist14. Therefore, alternative treatment methods are being sought, including those using natural plant compounds, including those derived from Chinese medicine. Their use in anticancer therapy is associated with their ease of acquisition and beneficial therapeutic effects, with minimal side effects14,15.
One such group of plant compounds are anthraquinones, belonging to aromatic compounds constituting the largest group of natural and synthetic quinones. They are compounds found in various plants, as exemplified by rhubarb and aloe, used for over 4000 years in folk medicine as laxatives. They are common in the human diet, used in food, cosmetics and pharmaceutical products6,16,17. They also have various effects, including anticancer, antibacterial, antioxidant, immunoregulatory, antihyperlipidemic, and their biological activity depends on the substitution of their hydroxyl groups in the anthraquinone ring structure18,19. Current therapeutic indications for the use of anthraquinones in pharmacology include the treatment of constipation, arthritis, multiple sclerosis, and cancer17. Their mechanism of anticancer action includes, among others, inhibition of proliferation, invasion, migration, and metastasis, and induction of apoptosis of cancer cells20. Our previous studies have shown that anthraquinones such as quinalizarin21, chrysophanol22, physcion23, rhein24, emodin25,26, and aloe-emodin27 exhibit anticancer effects also associated with the modulation of the lysosomal system, lysosomal cell death, and induction of mitotic catastrophe.
In the presented studies, we focused on alizarin, which showed multidirectional biological activity against cervical cancer and prostate cancer cells. Its mechanism of anti-cancer action was expressed through the induction of apoptotic processes, as indicated by the activation of executive caspases 3/7, inhibition of the anti-apoptotic protein Bcl-2 and DNA fragmentation (activation of ATM and H2A.X) (Figs. 1, 2).
In addition, to confirm the cytotoxicity of alizarin, we conducted studies on a 3D model, which can be used in the process of developing new anticancer drugs28. According to literature data, cancer cells often show lower sensitivity to chemotherapeutics in solid tumor models such as cell spheroids compared to monolayer cells in 2D culture. Spheroids with a diameter greater than 500 μm were used for the studies because they are believed to mimic the pathophysiological conditions prevailing in solid tumors, including a central hypoxic area and a proliferation gradient29,30. These properties give the spheroids a profile of resistance to anticancer drugs that is similar to that shown by human solid tumors28. Using fluorescence analysis, we confirmed that alizarin exhibited cytotoxic activity not only in the 2D model but also in the 3D model. This was evidenced by the presence of structurally damaged spheroids and numerous dead cells, as well as a high level of apoptotic cells revealed by flow cytometry analysis (Fig. 1), which was slightly lower than in the 2D culture.
Apoptosis is a characteristic type of cell death, which is induced by numerous chemotherapeutics in oncological therapy. However, during tumor progression, the homeostasis between anti- and pro-apoptotic regulators is disturbed. This applies especially to the high level of anti-apoptotic proteins, which is one of the main mechanisms allowing cancer cells to acquire resistance to chemo- and radiotherapy31. In our studies, we have also shown that the combination of Venetoclax—a selective inhibitor of the anti-apoptotic protein Bcl-2 with alizarin enhances its pro-apoptotic effect, which indicates their synergism in action. Combination therapy is a treatment method in which two or more therapeutic agents are combined, and the combination of anticancer drugs increases the effectiveness of treatment compared to monotherapy, which in turn may reduce drug resistance, reduce tumor growth and metastatic potential, and induce apoptosis32. This is especially important in relation to prostate cancer cells because currently attention is being paid to the possibility of using Bcl-2 inhibitors in the therapy of prostate cancer33. It is believed that apoptosis in prostate cancer cells can be restored by counteracting the excess of pro-survival proteins using BH3 mimetic drugs, which mimic the action of natural antagonists. In vitro studies on prostate cancer cell lines have shown that the above-mentioned drugs disrupt the interaction of pro- and antiapoptotic proteins from the BCL-2 family, and by sensitizing cells to the action of enzalutamide, they intensify the apoptosis of CRPC and LNCaP cells10. We also found that a greater cytotoxic effect occurs in the case of Venetoclax with alizarin in the tested cells, especially in prostate cancer cells, than in the case of their encumbrance with alizarin alone (Fig. 7).
Also in the case of cervical cancer, combined chemotherapy is used, which is the recommended strategy in treating the first throw. Frequent drugs used in combined therapy are cisplatin in combination with paclitaxel, topotekan and gemcitabine34,35. Studies by Karademir and Ozgur demonstrated that the combination of Ganetespib (an inhibitor of STA-9090, heat shock protein 90 (HSP90)) and Venetoclax significantly enhanced the cytotoxic effect and apoptosis in cervical cancer cells compared to Ganetespib alone. Inhibition of proliferation in the tested cells was also observed in the case of synergistic action of Venetoclax with Ganetespib9.
When analyzing problems related to anticancer therapy, attention was also drawn to the induction of oxidative stress associated with increased ROS levels, which consequently leads to disruption of redox homeostasis and cancer cell damage36. Prooxidant factors may be anticancer drugs, including Bortezomib (a proteasome inhibitor)37, doxorubicin, daunorubicin, or epirubicin38. Anthraquinones also exhibit proapoptotic activity, including alizarin, which generates reactive oxygen species (Fig. 3), which may be related to the progressive mitochondrial damage and increased endoplasmic reticulum stress observed in electron microscopy in the tested cells (Fig. 4). In the mitochondrial matrix vacuole-like structures were observed, a direct result of osmotic swelling of mitochondria in response to oxidative stress. Alizarin treatment intensievied the generation of reactive oxygen species and the destabilization of the mitochondrial membrane potential, which exacerbated the progression of changes toward apoptosis. Studies have shown that excessive production of reactive oxygen species was associated with the loss of mitochondrial membrane potential and accelerated progression of apoptosis, which is consistent with the involvement of mitochondria in apoptosis signaling pathways39,40. Therefore, it can be assumed that this type of vacuolization is a morphological expression of the physical breakdown of the mitochondrial matrix organization due to the loss of mitochondrial membrane integrity, which simultaneously highlights the effect of the tested anthraquinone on mitochondrial damage, which may correlate with the level of generated ROS.
The antiproliferative effect of chemotherapeutics is also very important in anticancer therapy. Alizarin also has such properties, which we demonstrated in the wound healing test and the clonogenic test. The tested anthraquinone also affected the blocking of cells in the G2/M phase of the cell cycle, which makes it similar to other phase-specific chemotherapeutics (Fig. 6). Cell cycle blocking agents are crucial for improving cancer treatment, as cell cycle regulation is associated with the occurrence, development, and metastasis of cancer. The regulation of the cell cycle in combination with chemotherapeutics may therefore constitute a current strategy for oncological therapy41.
In the case of cancer cell resistance to treatment, the induction of additional, alternative mechanisms of cell death, such as a mitotic death, is also crucial. Currently, mitotic catastrophe is considered a new and effective mode of cancer cell death in response to various anticancer drugs, including microtubule-targeting drugs, DNA-damaging drugs, or DDR inhibitors42. The mitotic catastrophe observed in our study was manifested by a significant increase in mitotic markers, such as giant cells, cells with micronuclei, and abnormal mitotic figures (Fig. 5). Numerous studies show that the effectiveness of anticancer therapy can also be achieved by several types of cell death, which include the induction of apoptosis, autophagy, senescence, or mitotic catastrophe, which can occur simultaneously in a complementary or competitive mode during cancer treatment42. It has been shown that long-term stress and sustained autophagy induced by anticancer drugs can ultimately lead to cancer cell death. It has been shown that increased autophagy due to the use of anticancer drugs causes autophagy-dependent cancer cell death43. As documented in Fig. 4, our study results showed that alizarin, in addition to apoptosis, induced in the studied cells also autophagic processes, which were manifested by the presence of numerous lysosomes, autophagosomes, and autophagolysosomes indicating increased cellular degradation. It is important to emphasize that the ultrastructural changes demonstrated correlated with alizarin’s modulating effect on LC3-II protein level (Supplementary Fig. S2). At the same time, a modulating effect of the applied alizarin concentrations on vacuolar changes was observed, as a gradual switch from autophagic processes towards apoptosis was observed with increasing concentration (Fig. 5). These observations further support the cytotoxic activity of alizarin toward cervical and prostate cancer cell models. Although alizarin treatment was associated with changes in autophagy-related markers, this study does not definitively determine whether autophagy acts cytoprotectively or prolethally. The simultaneous presence of autophagy-lysosomal structures and apoptotic features reflects the coexistence of these processes but does not constitute evidence of autophagy-dependent cell death. Furthermore, the decreased LC3-II signal observed at higher alizarin concentrations may indicate reduced autophagy initiation accompanying enhanced apoptosis progression rather than increased autophagic flux. Elucidating the function of autophagy would require pharmacological inhibition of late stages of autophagy, which was beyond the scope of this study.
The PI3K/AKT signaling pathway also plays a key role in various cellular processes, including proliferation, apoptosis, and drug resistance, and is abnormally activated in various types of cancer, contributing to disease progression44,45. PI3K/AKT signaling blocks the expression of proapoptotic proteins, reduces tissue apoptosis, and increases cancer cell survival46. Activation of the pathway has also been shown to be associated with the development of treatment resistance. Therefore, numerous inhibitors of the PI3K/AKT signaling pathway are currently being developed, and cancer treatment strategies including the use of combination therapy, i.e., pathway inhibitors with other drugs or therapies to improve treatment outcomes45. The MAPK/ERK pathway, whose dysregulation is observed in various types of cancer, also plays a significant role in cancer cell proliferation, survival, and invasion47. Numerous studies also indicate that these pathways interact, and enhancing one signaling pathway can enhance or inhibit another45.
Stimulation of PI3K/AKT signaling also promotes prostate tumor cell survival, prevents apoptosis, increases cell cycle progression, proliferation rate, and metastasis. This signaling pathway also mediates drug resistance and reduces the radiosensitivity of prostate tumor cells. Silencing PI3K/AKT signaling therefore impairs the growth and metastasis of prostate tumor cells44.
In cervical cancer, the PI3K pathway is activated by genetic mutations (in the PIK3CA gene), leading to uncontrolled cell growth, proliferation, and resistance to apoptosis. It is also activated by signals from human papillomavirus (HPV) and other receptors, contributing to the development and progression of cancer. This hyperactivation makes this pathway a key target for new therapies, and inhibitors are currently being sought that could inhibit its function and induce cell death48.
Our experiments show that alizarin significantly altered the activation of the PI3K/MAPK pathways in the studied cells, as evidenced by significantly reduced levels of pAkt and pERK and an increased number of double-negative cells (pAkt⁻/pERK⁻), with a concomitant increase in apoptosis (Supplementary Figure S3). Venetoclax (4 µM) also showed a moderate effect of dual phosphorylation of Akt and ERK, but also a transient increase in ERK activation (pERK⁺/pAkt⁻), particularly in the DU145 cell line, which decreased after the higher concentration (8 µM). Following combined treatment with alizarin and Venetoclax, a reduced proportion of MAPK- and PI3K-activated cell populations was observed, reflected by a greater than 80% increase in double-negative cells (pAkt⁻/pERK⁻). These changes correlated with an increased proportion of cells exhibiting Bcl-2 phosphorylation and with a significant increase in apoptosis in both HeLa and DU145 cell lines.
The combined treatment with alizarin and Venetoclax resulted in stronger cellular responses than alizarin alone across multiple experimental readouts. Quantitative analyses demonstrated that these differences were statistically significant when directly comparing single-agent and combined treatments. At the same time, the observed changes in MAPK and PI3K pathway-related cell populations reflect shifts in the distribution of signaling states rather than direct evidence of pathway inhibition. Together, these findings support the enhanced cellular impact of the combined regimen while remaining consistent with the descriptive nature of the pathway activation analysis.
These results suggest that alterations in PI3K/MAPK signaling accompany the proapoptotic effects of alizarin, particularly when combined with Venetoclax. The presented results support further investigation of alizarin in combination with Venetoclax in experimental cancer models, particularly in light of the ERK activation observed at lower Venetoclax concentrations, a signaling response that has been implicated in adaptive resistance mechanisms in prostate cancer cells.
The cytotoxic effects of alizarin and Venetoclax observed in 2D culture were also confirmed in studies of spheroids obtained from HeLa and DU145 cell lines (Fig. 8). The tested factors, especially in combination, were shown to modify the structure of spheroids, resulting in a loss of their integrity, which correlated with high levels of apoptosis, and such changes are often observed in studies of compounds with anticancer potential (Fig. 9).

Conclusions
In conclusion, the present study shows that alizarin affects multiple cellular pathways in cervical and prostate cancer cell models cultured in two- and three-dimensional systems. Alizarin treatment was associated with apoptotic features, increased caspase-3/7 activity, phosphorylation of Bcl-2, activation of DNA damage response markers, alterations in mitochondrial morphology accompanied by increased reactive oxygen species generation, inhibition of the PI3K/MAPK signaling pathway, and features consistent with mitotic catastrophe, along with modulation of autophagy-related processes. Moreover, combined treatment with alizarin and the selective Bcl-2 inhibitor venetoclax enhanced cytotoxic effects compared with single-agent treatment. Overall, these findings indicate that alizarin may be of interest in the context of combination anticancer strategies; however, further studies are required to clarify the underlying molecular mechanisms.

Material and methods

In vitro culture conditions
HeLa (cervical cancer), DU145 (prostate cancer) and VH10 (normal human fibroblasts) cells were cultured in DMEM (GIBCO, New York, USA) supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France) and a mixture of antibiotics (amphotericin B, penicillin G, streptomycin) (Corning, Manassas, USA) in a DirectHeat CO2 incubator (Thermo Fisher Scientific, Waltham, MA, USA). Cells were incubated for 48 h with alizarin (C14H8O4, ≥ 95.0% (HPLC)) (Sigma-Aldrich, St. Louis, MO, USA) at concentrations of 150 µM, 200 µM, 250 µM and 350 µM. Additionally, Venetoclax (ABT-199) (Selleck Biotechnology GmbH, Cologne) was used at a concentration of 4 µM and 8 µM to demonstrate synergistic action with alizarin (250 µM).

Generation of HeLa and DU145 spheroids model-Hanging drop method
Generation of HeLa and DU145 cell spheroids was performed using the hanging drop technique. For this purpose, 20 µl drops containing culture medium with cells were placed on the inside of the lid of a 35 mm diameter culture dish. After the lid was inverted, the surface tension is responsible for keeping the drops in place, and the microgravity inside each drop causes the cells to concentrate and generate spheroids. 1 ml of sterile PBS was added to the bottom of the culture vessel to create a hydration chamber for the cells. The dishes with hanging drops were placed in an incubator at 37 °C with 5% CO2 for 4 days. After the spheroids were formed, their growth was monitored by measuring their diameter (in 50 spheroids) using Nikon Nis Elements ER software, and the uniform spheroids were used for further studies. For this purpose, the culture medium was carefully removed, replaced with a new medium supplemented with 350 µM alizarin and alizarin combined with Venetoclax (ALIZ 250 µM + VENET 4 and 8 µM) and incubated for 48 h (analogously to the conditions in 2D culture).

Cell viability assessment-MTT test and FDA/PI staining
The MTT test is based on the reduction of the yellow MTT dye to purple formazan crystals by mitochondrial dehydrogenase only in living cells. After 48 h incubation with alizarin, cells were incubated for 2 h with MTT solution (1 mg/ml) (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide) (Sigma Aldrich, St. Louis, MO, USA) in 96-well plates (Falcon). The resulting formazan crystals were dissolved in DMSO (Sigma Aldrich, St. Louis, MO, USA), and the absorbance of the obtained solutions was read using a Synergy 2 microplate reader (BioTek, Winooski, VT, USA) at 570 nm. Additionally, cell viability was analyzed using double staining with fluorescein diacetate and propidium iodide (FDA/PI) according to the principle that fluorescein diacetate stains only living cells (conversion of fluorescein diacetate to fluorescein), while dead cells are stained with propidium iodide (penetration of the fluorochrome into the cell through the damaged membrane and binding to DNA). For this purpose, cells (2 × 104) after 48-h incubation with alizarin (350 µM) were trypsinized, centrifuged, and the cell pellet was stained using fluorescein diacetate (1 mg/ml) and propidium iodide (1 mg/ml) (Sigma-Aldrich, St. Louis, MO, USA). The stained cells were applied to a glass slide and analyzed microscopically using a Nikon 80i epi-fluorescence microscope (Nikon Instruments, USA). The number of stained cells was counted in 30 random fields using a 20 × objective lens, and then the average values were taken from the obtained results. The experiment was repeated 3 times.
Staining of cell spheroids. For this purpose, FDA/PI dyes were added to spheroids formed by the hanging drop technique and incubated for 10 min at 37°C. Immediately after staining, the spheroids were analyzed under a Nikon Ti inverted fluorescence microscope using fluorescence filters corresponding to the dyes used (FDA-480 nm, excitation; 520 nm, emission; PI-535 nm, excitation; > 610 nm, emission) (Nikon Instruments, USA). The experiment was repeated 3 times.

Detection of apoptosis-Annexin V assay
The percentage of apoptotic cells was assessed using the Annexin V Dead Cell Kit (Merck KGaA, Darmstadt, Germany). After 48 h of incubation with alizarin, cells were trypsinized (0.25% trypsin–EDTA solution) (Corning, Manassas, USA), centrifuged and stained (100 µl Annexin V-PE/7-AAD) for 20 min in the dark at room temperature. Fluorescence intensity was read on a Muse analyzer (Merck-Millipore, USA). The experiment was repeated 3 times.
The Annexin V assay was also used to quantify dead cells in the spheroid structure. For this purpose, the spheroids formed in each hanging drop were collected in an Eppendorf tube. The spheroids were broken by mechanical pipetting, and the single cells obtained in this way were stained with Annexin V according to the above-mentioned methodology.

Caspase 3/7 activity
The percentage of caspase-positive cells was determined using a Muse analyzer (Merck-Millipore, Guyancourt, France) using the Muse Caspase-3/7 Kit (Merck-Millipore, Guyancourt, France). After 48 h of incubation with alizarin, the cells were trypsinized, centrifuged, and the resulting cell pellet was incubated for 30 min at 37 °C with 5 µl of Caspase-3/7 working solution. The experiment was repeated 3 times.

Assessment of Bcl-2 protein phosphorylation
Changes in Bcl-2 phosphorylation in the studied cells were assessed using the Muse Bcl-2 Activation Dual Detection Kit (Merck-Millipore, Guyancourt, France). In the assay, two directly conjugated antibodies were used to assess the level of Bcl-2 expression, i.e. phosphospecific anti-phospho-Bcl-2 (Ser70)-Alexa Fluor®555 and anti-Bcl-2-PECy5. The degree of activation of the Bcl-2 pathway was performed by measuring Bcl-2 phosphorylation in relation to the total expression of Bcl-2 in the studied cells.

Dual PI3K/MAPK Pathway Activation Assay
The Muse® Dual PI3K/MAPK Pathway Activation Kit (Merck-Millipore, Guyancourt, France) was used in this experiment to simultaneously assay the PI3K and MAPK signaling pathways using the Muse Cell Analyzer (Merck-Millipore®, Burlington, MA, USA). The kit contains two directly conjugated antibodies: a phosphospecific Akt (Ser473)-Alexa Fluor™ 555 antibody and a phosphospecific ERK1/2 (Thr202/Tyr204, Thr185/Tyr187)-PECy5 conjugated antibody, which enable the simultaneous assessment of PI3K and MAPK signaling pathway activation. For this purpose, after 48 h of incubation with 250 µM alizarin and 250 µM alizarin with 4 and 8 µM Venetoclax, cells were fixed with fixative solution, followed by cell permeabilization buffer (10 min on ice). Antibody solution (10 µL) was then added to the cell suspension from each treatment group and incubated for 30 min in the dark at room temperature. Afterwards, cells were washed in buffer and analyzed.

Demonstration of synergistic activity of alizarin with Venetoclax
A concentration of 250 µM was used to determine the synergistic effect of alizarin with Venetoclax (4 and 8 µM), resulting in an approximate IC50 value in the MTT assay. The combined effect was assessed using the MTT reduction assay and flow cytometry, which allowed for the determination of the level of apoptosis, including the percentage of caspase-positive cells, Bcl-2 protein levels, and PI3K/MAPK pathway activity.

DNA damage assessment
After 48 h of alizarin treatment, cells were fixed and permeabilized using reagents from the Muse Multi-Color DNA Damage kit (Merck-Millipore, Guyancourt, France) to assess the percentage of double-stranded DNA breaks. Then, cells were stained with anti-phospho-Histone H2A.X (Ser139) and anti-phospho-ATM (Ser1981). Using the kit, the percentage of negative cells (without DNA damage), the percentage of cells with activated ATM and H2A.X and with double DNA breaks (double activation of ATM and H2A.X) were determined.

Measurement of reactive oxygen species production
Muse Oxidative Stress Kit (Merck Millipore Guyancourt, France) was used to assess oxidative stress. For this purpose, cells after 48 h of alizarin treatment were treated with Muse Oxidative Stress Reagent working solution (190 µl) and then incubated for 30 min at 37 °C. Based on the flow cytometric analysis, the percentage of ROS (−) and ROS (+) cells was determined. The experiment was repeated 3 times.

Mitochondrial membrane potential (Δψm) measurement
The decrease in Δψm was analyzed using the Muse Mitopotential Assay kit (Merck Millipore, Guyancourt, France). After alizarin treatment, cells were added to MitoPotential working solution and incubated for 20 min at 37 °C. Then, cells were stained with 7-AAD (5 µL) at room temperature for 5 min and analyzed on the analyzer. The experiment was repeated 3 times.

Ultrastructure analysis
Cell fixation was performed in 3% glutaraldehyde in 0.1 M cacodylate buffer, pH = 7.3 (Serva Electrophoresis GmbH, Germany), and secondary fixation in 2% osmium tetroxide (Spi, West Chester, PA, USA). Then, the cells were dehydrated in ethyl alcohol (10–99.8%), embedded in Epon 812 epoxy resin (Serva Electrophoresis GmbH, Germany) and polymerized at 40 and 60 °C. Ultrathin sections cut on a Leica EM UC7 ultramicrotome (Leica Biosystems, Germany) and contrasted with uranium acetate and lead citrate were analyzed using a Tecnai G2 Spirit transmission electron microscope (FEI, Company USA) equipped with a Morada camera (Olympus, Soft Imagine Solutions, Münster). Mitochondria were also quantified and measured using TEM Imaging & Analysis 3.2 SP6 software (FEI Company, Hillsboro, OR, USA). Mitochondrial length was demonstrated by measuring organelles in 100 cells from the control and alizarin-treated groups. Mean values were determined from the obtained results.

Assessment of LC3-II antibody levels
The LC3 antibody detection assay (Merck Millipore) was used to assess autophagy levels. According to the assay principle, cytosolic LC3 (LC3-I) is conjugated with phosphatidylethanolamine to form an LC3-phosphatidylethanolamine (LC3-II) conjugate recruited to autophagosome membranes. LC3-II is degraded in the lumen of lysosomes. The turnover of the lysosomal autophagosomal marker LC3-II reflects autophagic activity, and its level increases after the induction of autophagosome formation, but then decreases after the fusion of autophagosomes with lysosomes. According to the assay instructions, cells were seeded on 96-well plates. After 24 h, cells were incubated with alizarin. After 48 h, the cells were added to selective membrane permeabilization reagent (Autophagy Reagent A) in Earle’s salt solution (EBSS medium) and incubated for 4 h. The reagent allows for the differentiation of cytosolic and autophagic LC3, which is possible due to the extraction of cytosolic protein while protecting LC3-II, which is translocated to autophagosomes and remains intact there. Then, the cells were washed with HBSS, trypsinized, centrifuged, the supernatant removed, and anti-LC3 Alexa Fluor® 555 and Autophagy Reagent B were added to the cells and incubated in the dark on ice for 30 min. The samples were then centrifuged and analyzed by flow cytometry. The positive control consisted of cells incubated for 4 h in serum-free medium. The experiment was performed in triplicate.

Hematoxylin–eosin (H&E) staining—assessment of morphological changes
Cells cultured on sterile coverslips in dishes (Falcon) were fixed in methanol, stained with Harris hematoxylin and eosin (Sigma Aldrich, St. Louis, MO, USA), dehydrated in an ascending alcohol series and exposed in xylene. Morphological analysis was performed using a Nikon Eclipse 80i microscope with digital image analysis software (Nikon NIS Elements D 3.10) (Nikon Instruments, Inc., NY, USA). In preparations, 3,000 cells were analyzed in three independent experiments (9,000 cells/concentration). Mitotic catastrophe was determined on the basis of morphological indices such as giant cells, multinucleated cells, cells with micronuclei and the presence of abnormal mitotic figures.

Cell cycle analysis
After 48 h of incubation with alizarin, they were fixed in ice-cold 70% ethanol and then a cell cycle assay kit (Merck-Millipore, Guyancourt, France) based on a nuclear DNA intercalating dye, propidium iodide (PI) and RNAse A was used. The stained cells were analyzed using a Muse analyzer (Merck-Millipore, Guyancourt, France) to determine the percentage of cells in the individual cycle phases. The experiment was performed in triplicate.

Cell migration assay-Scratch wound healing assay
The degree of cell migration inhibition was analyzed using a scratch wound healing assay. Cells were cultured in growth medium for 24 h to form a confluent monolayer. A linear scratch was generated in each well using a sterile 10-µL pipette tip. The wells were washed with PBS to remove cell debris, and fresh medium containing alizarin (150–350 µM) was added; control cells were treated with medium alone. After 24 and 48 h of incubation, wound images were acquired under phase-contrast conditions using a Nikon Eclipse Ti inverted microscope (Nikon Instruments Inc.) equipped with a cell culture system (Okolab) consisting of an incubation chamber, a humidity module, and a gas mixer. Cell migration was quantified by measuring the distance between the wound edges at each time point using NIS-Elements AR image analysis software (Nikon Instruments Inc.). Cell migration (%) was calculated as the percentage of wound closure relative to the initial wound width (0 h). Data are presented as mean values.

Cell migration was assessed using a scratch wound healing assay.
Cells were seeded in growth medium and cultured for 24 h to form a confluent monolayer. A linear scratch was generated in each well using a sterile 10-µL pipette tip. After washing with PBS to remove detached cells, fresh medium containing alizarin (150–350 µM) was added; control cells received medium without alizarin. Images were acquired immediately after scratching (0 h) and after 24 and 48 h of incubation using a Nikon Eclipse Ti inverted phase-contrast microscope (Nikon Instruments Inc.) equipped with an environmental control system (Okolab). Cell migration was quantified by measuring the wound width at each time point using NIS-Elements AR image analysis software (Nikon Instruments Inc.). Cell migration (%) was calculated as the percentage of wound closure relative to the initial wound width (0 h) and normalized to the control group. Data are presented as mean values.

Clonogenic assay
The clonogenic capacity of the tested cells after exposed to alizarin was assessed using a colony formation assay. HeLa and DU145 cells were seeded on culture dishes (100–600 cells/plate) and cultured for 24 h. Then, the cells were treated with different concentrations of alizarin (150–350 µM). After 14 days of incubation, the medium was removed, and the resulting cell colonies were fixed in 3.7% paraformaldehyde for 30 min and stained with 0.05% crystal violet. Colonies containing more than 30 cells were counted. Clonogenic survival was expressed as survival (%), calculated as the number of colonies formed after treatment relative to the untreated control, which was set to 100%. Data are presented as mean values.

Statistical analysis
The analysis of the results was performed using one-way analysis of variance (ANOVA), with post-hoc multiple comparisons using Tukey’s test. P < 0.05 was considered statistically significant. Statistica 13.3 software (StatSoft, Poland) was used for data analysis.

Drug Interaction Analysis (Bliss Independence Model)
The effects of drug combinations were analyzed using the Bliss independence model. The expected fractional effect (Fa_expected) was calculated using the equation:

where Fa1 and Fa2 are the fractions of cells affected by each drug individually, determined from the MTT assay data. The observed fraction affected (Fa_observed) for each combination was compared to the Bliss predicted value. Calculations and data visualization were performed in Microsoft Excel (Microsoft Corp., Redmond, WA, USA) using manually entered formulas to ensure clarity and reproducibility.
Drug interactions were interpreted as synergistic when Faobserved > Faexpected, additive when Faobserved ≈ Faexpected, and antagonistic when Faobserved < Faexpected.

Supplementary Information