Cancer is currently a significant public health issue, ranking among the leading causes of death worldwide. Approximately 70% of cancer-related deaths occur in developing nations. According to estimates by the International Agency for Research on Cancer (IARC), the annual number of new cancer cases is projected to increase to around 21.7 million by 2030, with deaths reaching 13 million (Dalton et al., 2019). Among the various strategies for cancer treatment, chemotherapy is widely recognized as one of the most commonly used methods to prevent and treat cancer. However, current chemotherapeutic drugs have significant side effects and can lead to acquired drug resistance (Srivastava et al., 2016). Therefore, it is imperative to identify new, effective, and less toxic therapeutic agents for the treatment of cancer. Plants possess considerable potential for the provision of chemotherapeutic effects against cancer. Over 60% of anti-cancer drugs have been derived from natural sources, including plants, microorganisms, and marine organisms (Dey et al., 2019). The research presented in this work makes use of a number of substances derived from natural sources. Biochanin A (5,7-dihydroxy-4′-methoxyisoflavone) is an isoflavone that can be found, among others, in red clover (Trifolium pratense L.) (Yu et al., 2019). Biochanin A has been demonstrated to inhibit epithelial-mesenchymal transition and to reduce the proliferation rate of lung cancer cells. This is achieved by activating the Bcl-2 and caspase-3 pathways, in addition to regulating the expression of cell cycle-related proteins (Y. Li et al., 2018). The polyphenol compound curcumin, derived from the spice turmeric (Curcuma longa), has been demonstrated to inhibit the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling pathway in a range of cancer types, including rhabdomyosarcoma. As a naturally occurring chemotherapeutic agent, curcumin has the potential to prevent and slow the progression of carcinogenesis. Additionally, it exhibits antioxidant and anti-inflammatory properties. The inhibition of factors responsible for cell proliferation and the inhibition of apoptosis, such as COX-2, AP-1, and NF-κB, is likely to occur as a result of the prevention of IκB phosphorylation by curcumin (Radzka et al., 2023). Berberine is a compound belonging to the isoquinoline alkaloid group that occurs naturally in a number of plant species, including those belonging to the Annonaceae, Berberidaceae, and Menispermaceae genera (Neag et al., 2018). Berberine exerts its effect by inhibiting the phosphorylation of the nuclear factor IκBα, which is responsible for activating NF-κB. Furthermore, evidence indicates that berberine treatment impairs the phosphorylation of the NF-κB subunit transcription factor p65. Berberine has been demonstrated to possess anti-cancer properties, whereby it downregulates caspase-1/IL-1β and also inhibits the cell cycle at the G1 phase in RMS cells (Jin et al., 2017). Caffeic acid phenethyl ester (CAPE) is a component identified in propolis. A substantial body of scientific literature attests to the antioxidant, anti-inflammatory, and cytotoxic effects of CAPE on cancer cells. It has been demonstrated that CAPE can inhibit NF-κB signaling by preventing the activation of upstream kinases, including IκB kinase (IKK) and TGF-β-activated kinase (TAK1), which are involved in the activation of the NF-κB pathway (Wang et al., 2010). Furthermore, evidence indicates that caffeic acid can inhibit the nuclear translocation of NF-κB and its binding to DNA, which are crucial steps in the transcriptional activation of NF-κB target genes (Abdel-Latif et al., 2005). Cucurbitacin E (CurE) is a triterpenoid compound that has been identified in a number of different plant species, including bitter melon (Momordica charantia) and cucumber (Cucumis sativus) (Chanda et al., 2020). This compound has been shown to modulate NF-κB signaling by inhibiting its activation and nuclear translocation (Qiao et al., 2013). Cucurbitacin E has been demonstrated to inhibit TNF-α-induced phosphorylation of inflammatory cytokines by modulating the NF-κB pathway. Additionally, cucurbitacin E inhibits the PI3K/Akt/mTOR pathway and the epithelial-mesenchymal transition (EMT) in osteosarcoma cells (Jia et al., 2015).

The NF-κB factor has been identified as the regulator of genes involved in tumour promotion, including those related to cell proliferation, angiogenesis, and adhesion. Furthermore, the activation of NF-κB has been demonstrated to influence the production of prostaglandins through the COX2 protein, which is overexpressed in a number of different types of cancer (Colotta et al., 2009). In neoplastic tissues, where elevated levels of the NF-κB factor are observed, there is an accumulation of pro-inflammatory cytokines, which contribute to the creation of a pro-neoplastic environment. Prolonged inflammation results in genomic instability and the emergence of genetic mutations that promote the formation and development of tumours (Elinav et al., 2013). Fibrosarcoma, an aggressive and poorly understood form of soft tissue sarcoma, poses significant therapeutic challenges, and recent evidence suggests that the NF-κB pathway may play a key role in its progression or angiogenesis (46). In this study, we demonstrated the effects of naturally occurring NF-κB inhibitors on fibrosarcoma cells, a cancer type where NF-κB’s role has not been extensively characterised in the context of these specific inhibitors. Although previous studies have investigated the activity of these compounds in various cancer models, their effects on fibrosarcoma remain underexplored, representing an important area of investigation. Fibrosarcoma is a particularly aggressive form of soft tissue sarcoma with a paucity of efficacious therapeutic options. Emerging evidence suggests that the NF-κB pathway plays a crucial role in its progression. By targeting NF-κB in fibrosarcoma, our objective is to provide new insights into potential therapeutic approaches for this malignancy. Apoptotic genes, including FLICE inhibitory protein, survivin, XIAP, c-IAP1/2 inhibitor, and the Bcl-2 family of proteins, are overexpressed in numerous types of cancer cells. The NF-κB factor induces this overexpression. It is therefore postulated that NF-κB plays a role in the regulation of anti-apoptotic mechanisms in the context of tumour formation (Kucharczak et al., 2003). A study of human prostate cancer cells (PC-3M cell line) has demonstrated that NF-κB plays a role in angiogenesis, invasion, and metastasis by regulating vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP) (Huang et al., 2001). It has been demonstrated that the NF-κB factor exerts an influence on the resistance of cells to a range of chemotherapeutic agents. NF-κB plays a role in regulating the expression of the mdr1 gene, which is responsible for producing P-glycoprotein (P-gp). P-glycoprotein is a plasma membrane-associated multidrug transporter that affects the efflux of chemotherapy drugs, thereby contributing to treatment resistance (Wang et al., 2015). It has been demonstrated that NF-κB inhibits P-glycoprotein expression and may reactivate chemosensitivity. The involvement of the NF-κB factor in the degradation of muscle proteins has been demonstrated by research using cell cultures and in in vivo studies on laboratory animals (Wyke et al., 2004). In the study conducted by Moore–Carrasco et al., rats with hepatocellular carcinoma (AH-130 cell line) were treated with an NF-κB inhibitor (SP100030) and an activator protein-1 (AP-1) activator. The treatment was found to be effective in addressing skeletal muscle atrophy (Moore-Carrasco et al., 2007). Muscle wasting is a common occurrence in individuals of advanced age and those suffering from pathological conditions such as cancer. A study using a murine model indicated that the levels of NF-κB were elevated in the skeletal muscle tissues of animals with Lewis Lung Cancer (LLC). The study demonstrated that the transgenic overexpression of the super-repressor mutant protein IκBα (IκBαSR) resulted in a significant reduction in skeletal muscle and body weight loss in mice with LLC, which was achieved by inhibiting NF-κB activity (Cai et al., 2004). Recently, there has been a surge in interest in fructose 1,6-bisphosphatase (FBP) in the scientific community. This is a consequence of research which has demonstrated that FBP not only regulates the synthesis of glucose and glycogen from carbohydrate precursors, but also impacts (in a non-catalytic manner) a variety of cellular processes thanks to interactions with numerous proteins, e.g., mitochondrial VDAC, ANT, and ATP synthase, CAMK2, and transcription factors HIF1α and NF-κB. As a result of these interactions, FBP exerts influence over a number of cellular processes, including cell cycle-related events, mitochondrial biogenesis and membrane polarization, microtubule stability, glycolytic enzyme expression, synaptic plasticity, and even the progression of cancer (Gizak et al., 2012; Pirog et al., 2014). Mammalian tissues exhibit two distinct isoenzymes of fructose 1,6-bisphosphatase: liver FBP (FBP1) and muscle FBP (FBP2). The physiological function of FBP2 is modulated not only by its cellular expression levels but also by its oligomeric state. The dimeric form of FBP2 is associated with mitochondria, where it confers protection against cellular stress, while the tetrameric form is localized within the cell nucleus (Wiśniewski et al., 2017). FBP exerts negative effects on cell growth, and inhibits the progression of gastric cancer (Li et al., 2013). A study conducted in 2016 assessed the transcriptional levels of FBP1 and FBP2, revealing that elevated levels of FBP1 mRNA were associated with a favorable prognosis in gastric cancer. Subsequent verification of FBP1 protein expression confirmed that its overexpression correlated with improved clinical outcomes in patients with gastric cancer following surgical resection. Functional analyses further demonstrated that FBP1 expression significantly inhibited the proliferation and invasion of gastric cancer cells (Li et al., 2016). Additionally, FBP2 has been demonstrated to impede the progression of sarcomas by inhibiting mitochondrial biogenesis. It has been demonstrated that FBP2 impedes the progression of sarcoma by hindering mitochondrial biogenesis. Soft tissue sarcomas (STS) constitute a heterogeneous group of neoplasms that originate from connective tissues. Despite the considerable genetic heterogeneity observed among these tumours, an aberrant glucose metabolism is a common feature, although the underlying mechanism remains elusive. As was reported, FBP2 is absent in numerous STS subtypes, and reintroduction of FBP2 has been observed to significantly slow the growth of sarcomas. The researchers identified two distinct tumour-suppressing roles for FBP2 based on its location within the cell. Cytosolic FBP2 was observed to decrease glucose breakdown through its enzymatic function, while nuclear FBP2 was found to suppress a crucial factor involved in mitochondrial formation and function. The two roles of FBP2 result in a reduction in the energy supply to the cancer cells, which provides an explanation for the frequent loss of FBP2 in STS and its potential as a therapeutic target (Huangyang et al., 2020). In 2022, studies demonstrated that the chemically induced tetramerization of FBP2, resulting in a reduction of FBP2-mitochondria interactions, was associated with a range of changes in HL-1 cells. These included a decrease in mitochondrial membrane potential, disruption of the tubulin network and tubulin-mitochondria interactions, a marked reduction in mitochondrial membrane potential, and an increase in the speed and extent of mitophagy (Pietras et al., 2022). Furthermore, motor proteins, such as myosin, utilize ATP to generate force and facilitate movement along these actin filaments. This movement is essential for a number of cellular activities, including cell motility and structural integrity (Masters et al., 2017). The particular actions of myosins in relation to actin serve to illustrate their function in the organisation of the cytoskeleton and in the performance of cellular processes. Myosins, driven by the hydrolysis of ATP, interact with actin filaments, enabling their movement. This process is crucial for a range of cellular activities, including migration, division, and maintaining cellular shape (Nambiar et al., 2010). Recent research indicates that FBP2 is present in mitochondria under specific circumstances, where it interacts with a range of mitochondrial proteins. This mitochondrial localization is significant as it may affect processes such as fusion and fission, which are vital for maintaining the functionality and structural integrity of mitochondria. One investigation demonstrated that FBP2 interacts with proteins such as Voltage-Dependent Anion Channels (VDAC), Adenine Nucleotide Translocator (ANT), and elements of the mitochondrial ATP synthase complex. These interactions have the potential to influence mitochondrial membrane potential and regulate mitochondrial biogenesis. The presence of FBP2 in the mitochondria might also be involved in managing the mitochondrial response to stress, as well as processes such as mitochondrial trafficking and mitophagy (Collins, 2004). Furthermore, mitochondrial dynamics, which encompass the processes of fusion and fission, are vital for preserving mitochondrial structure, distribution, and functionality within cells. Fusion serves as a quality control mechanism, whereby the contents of partially damaged mitochondria are mixed. In contrast, fission is essential for the removal of damaged mitochondrial components and the distribution of mitochondria during cell division. The involvement of FBP2 in these processes indicates that it may have a broader role in cellular homeostasis and the response to metabolic changes (Reynolds, 1963). The results of our studies presented here support the hypothesis that natural compounds acting as NF-κB inhibitors may be effective anticancer agents, particularly in the treatment of fibrosarcoma.

Detection of DNA fragmentation associated with apoptosis or the intermediate damage neutral comet assay method described by Collins was used (Collins, 2004). Briefly, after exposure to the examined compounds, WEHI-164/L6 cells (104 cells/well) were subjected to a trypsinization process. In the next step, 1x105/ml cells were washed with PBS chilled to 4°C, mixed with low-temperature melting agarose at a ratio of 1:10, and spread on a slide glass. Slides were submerged in precooled lysis buffer (2.5 M NaCl, 100 mM EDTA, pH 10, 10 mM Tris base, and 1% Triton X-100) at 4°C for 1 h. After lysis and rinsing, slides were equilibrated in TBE solution (40 mM Tris/boric acid, 2 mM EDTA, pH 8.3) and electrophoresed at 1.0 V/cm for 20 min and then silver staining was performed. For scoring the comet pattern, 100-200 nuclei were counted from each slide. The ranking of apoptotic comets was performed using the method developed by Collins (Collins, 2004).

Ultrastructural analysis of L6 and WEHI-164 cell lines was performed. The cells were incubated for 24 h with cytotoxic substances: biochanin A (5 µM), CAPE (25 µM), CurE (2.5 µM), curcumin (20 µM) or berberine (50 µM). Untreated cells were used as a control. The cells were then fixed for 30 min in 2.5% (vol/vol) glutaraldehyde and 0.1M phosphate buffer (pH 7.4). After post-fixation in 1% (wt/vol) osmium tetroxide, cells were dehydrated through a graded series of acetone and embedded in Epon (Sigma Aldrich, St. Louis, MI, USA). The Epon blocks were cut on Reichert Ultracut E. Ultrathin sections were, contrasted with uranyl acetate and lead citrate according to the standard method (Kong et al., 2009) and examined with a Talos L120C (ThermoFisher).

MTT assay assessed the cytotoxicity of the biochanin A, CAPE, CurE, curcumin and berberine. Figure 1 shows the response of L6 cells to 24 h and 48 h incubation with the analyzed substances. Incubation with berberine (e) and CurE (d) did not significantly change the mitochondrial activity of L6 cells compared to the untreated control group. In the case of incubation of L6 cells with biochanin A (c) it was observed that the cytotoxic effect increased with increasing concentration of the substance, both in the case of 24 h and 48 h incubation. At the highest concentration of 30 μM, cell survival after 24 h of incubation was ∼30% and after 48 h–18%. Incubation of cells with curcumin (a) showed an enhanced cytotoxic effect with prolonged incubation time (48 h). After exposure of cells to CAPE (b), the strongest cytotoxic effect was observed at a concentration of 1 µM (48 h). Then, with increasing CAPE concentration and prolonged incubation (48 h), cell survival increased, which can be possibly induced by the decay of CAPE in cells.

Figure 1. L6 cells viability measured by the MTT assay after 24 h and 48 h incubation with (A) berberine, (B) biochanin A, (C) CurE, (D) curcumin, (E) CAPE. Notes: (mean ± SD) N = 3, *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 2 shows the response of WEHI-164 cells to 24 h and 48 h incubation with the analyzed substances. Control represents the viability of untreated cells. WEHI-164 cells showed increased sensitivity to all of the tested substances. In the case of incubation with berberine (e), the strongest cytotoxic effect was obtained after 48 h of incubation at a concentration of 10 μM, where the percentage of cell survival was ∼33%. There was a clear decrease in the mitochondrial activity of cells incubated with biochanin A (c) at a concentration of 10 μM–30 µM and was ∼17%–9% (24 h). When incubated with CurE (d) and curcumin (a), cell survival decreased significantly with prolonged incubation and increasing concentration. All analysed substances had a stronger effect on cancer cells from the WEHI-164 line than on normal muscle cells from the L6 line. Table 1 presents the IC50 values calculated for biochanin A, curcumin, berberine, CAPE and CurE.

Figure 2. WEHI-164 cells viability measured by the MTT assay after 24 h and 48 h incubation with (A) berberine, (B) biochanin A, (C) CurE, (D) curcumin, (E) CAPE. Notes: (mean ± SD) N = 3, *p < 0.05, **p < 0.01, ***p < 0.005

Table 1. IC50 values for the tested compounds determined after 24 h treatment of L6 and WEHI-164 cells.

The apoptotic cell death was detected by neutral comet assay. The obtained results are demonstrated in Figures 3 and 4. Apoptotic cells in the L6 line were observed after 24 h of incubation with curcumin (12.5%) and CurE (1%) (a). No cell death was observed after exposure to the remaining substances. After 48 h of incubation of the L6 cells, apoptosis was observed in the case of incubation with curcumin (15.8%), CAPE (13.3%), biochanin A (3.6%) and CurE (2.5%) (c). In the case of WEHI-164 cells, apoptotic cells were observed after 24 h of incubation with CAPE (3.5%) and berberine (2.5%) (b). After 48 h of incubation of the WEHI-164 cells, apoptosis was observed after incubation with CAPE (12.7%), curcumin (4.4%), CurE (3.1%), biochanin A (2.2%) and berberine (1.6%) (d). There was a significant difference in the number of cells indirectly damaged after an extended (48 h) incubation time with the analysed substances. In the case of L6 cells, the highest percentage of damaged cells was observed following incubation with berberine (53%), while in the case of WEHI-164 cells, the highest percentage of damaged cells was observed following incubation with biochanin A (89.5%).

Figure 3. The neutral comet assay was performed on L6 and WEHI-164 cells after 24 h (A, B) and 48 h (C, D) of incubation with berberine, biochanin A, CurE, curcumin or CAPE.

Figure 4. Example micrographs of assessed comets in not damaged, intermediately damaged and apoptotic states. Magnification ×600.

Immunocytochemical staining of PARP-1 and caspase-3 in L6 and WEHI-164 cells treated with biochanin A (5 µM), CAPE (25 µM), CurE (2.5 µM), curcumin (20 µM) or berberine (50 µM) was performed (Figures 5 and 6). PARP-1 is cleaved by caspase-3 during apoptosis, which leads to its inactivation (Wiśniewski et al., 2017), (Li et al., 2013). The results of immunocytochemical analyses of the apoptotic proteins are presented in Table 2. PARP-1 overexpression was observed in L6 cells after exposure to each compound in ca. 10%–15% of cells similarly to control cells. Caspase-3 expression was observed in ∼<5% of untreated L6 cells. In the case of incubation with biochanin A and berberine, the expression level of caspase-3 was increased in 45% of cells, and in other cases in 80%–90% of cells. PARP-1 overexpression was observed in WEHI-164 cells at a low level, i.e., in less than 5% of cells after berberine treatment and in untreated cells. After the exposure to biochanin A, CAPE and CurE, PARP-1 expression was increased in 40%–50% cells. The highest level of PARP-1 95% of cells, was noted after incubation with curcumin. Interestingly, in the case of control WEHI-164 cells caspase-3 expression was observed in 40% of cells, and after incubation with biochanin A, CAPE, CurE and curcumin it was detected in 85%–95% of cells. Only after the treatment with berberine, expression of caspase-3 was observed in less than 5% of cells.

Figure 5. Caspase-3 and PARP-1 expression in L6 cells after 24 h incubation with curcumin, CurE, CAPE, berberine and biochanin A.

Figure 6. Caspase-3 and PARP-1 expression in WEHI-164 cells after 24 h incubation with curcumin, CurE, CAPE, berberine and biochanin A 1.3. Molecular Docking.

Table 2. Evaluation of immunocytochemical reaction with anti-PARP-1 and anti-caspase 3 antibodies in L6 and WEHI-164 cells, after exposition to biochanin A (5 µM), CAPE (25 µM), CurE (2.5 µM), curcumin (20 µM) and berberine (50 µM) for 24 h

The obtained results indicate that CAPE, biochanin A and CurE showed anticancer activity (Figure 7). The compounds are also NF-κB and cell cycle inhibitors and therefore should be considered in the treatment of muscle cancers. According to molecular docking, the most likely binding site corresponds to the Taxotere (TMR) binding site (Dey et al., 2019). TMR is an agent that inhibitors actin polymerization, thus the same site of CAPE and biochanin A binding may indicate that the substances will extend similar effect on actin and tubulin.

Figure 7. Molecular docking of actin (PDB:1J6Z) and tubulin (PDB:1TUB) with CAPE, biochanin A and CurE.

Berberine does not act specifically on actin or tubulin but interacts with intracellular kinases (AMP-activated kinases). This is an important distinction because the results of molecular docking of berberine to actin and tubulin would not reflect the actual mechanism of action of this compound (Alakurtti et al., 2006).

Curcumin, the active component of turmeric, is known for its potential anti-cancer, anti-inflammatory, and antioxidant properties. However, its interactions with tubulin and actin, key cytoskeletal proteins, have not been extensively studied. This is due to the high binding specificity required by these proteins, which may not be achieved by curcumin’s broad spectrum of activity. Additionally, the dynamic nature of the cytoskeleton makes experimental confirmation of these interactions challenging. Focusing solely on docking with tubulin and actin may not fully reflect curcumin’s anti-cancer mechanism, considering its interactions with multiple signalling pathways and molecular targets. These factors limit the attractiveness of curcumin as a targeted therapeutic agent for cytoskeletal protein interactions.

In addition to NF-κB, we included tubulin as a secondary target in our molecular docking studies. Tubulin was selected due to its well-established role in cancer cell division and its relevance as a therapeutic target. Inhibiting tubulin can disrupt the mitotic process, which may enhance the anti-proliferative effects of NF-κB inhibition. Although this dual-target approach provides a promising strategy for cancer treatment, it should be noted that molecular docking, while useful for predicting potential binding interactions, offers only preliminary insights.

Transmission electron microscopy (TEM) was employed to investigate the ultrastructural effects of biochanin A, curcumin, and berberine on sarcoma (WEHI-164) and normal muscle (L6) cells. This analysis aimed to elucidate the cellular stress mechanisms and morphological changes induced by these natural compounds, providing insights into their potential anticancer activity. By comparing treated and untreated cells, we were able to discern distinct cytotoxic and protective effects, highlighting the differential responses of normal and cancerous cells to these agents. The results are shown in Figures 8–12 below.

Figure 8. Morphology of untreated cells, where (A, B) WEHI-164, and (C, D) L6 cells. IV–intracellular vesicles; LD–lipid droplets; mt–mitochondria; Nu–cell nucleus; RER–rough endoplasmic reticulum.

Figure 9. WEHI-164 cells’ morphology after the exposure to (A, B) CAPE (25 µM), (C, D) CurE (5 µM), (E, F) berberine (50 µM). AB–apoptotic bodies; IV–intracellular vesicles; mt–mitochondria; Nu–cell nucleus.

Figure 10. A comparative analysis of the morphological characteristics of WEHI-164 cells treated with (A, B) biochanin A (5 µM) and (C, D) curcumin (20 µM). IV–intracellular vesicles; LD–lipid droplets; mt–mitochondria; Nu–cell nucleus.

Figure 11. A comparison of the ultrastructure o L6 cells, treated by (A, B) CAPE (25 µM), (C, D) CurE (5 µM) and berberine (E, F) (50 µM). IV–intracellular vesicles; LD–lipid droplets; ly–lysosomes. mt–mitochondria; Nu–cell nucleus.

Figure 12. Ultrastructural effects of (A, B) biochanin A (5 µM) and (C, D) curcumin (20 µM) on L6 cells. AB–apoptotic bodies. IV–intracellular vesicles. mt–mitochondria. Nu–cell nucleus.

Figure 8 shows that cells typically exhibit a particularly dark cytoplasm, a regular cell membrane, a barely discernible cell nucleus (Nu), a well-developed rough endoplasmic reticulum (RER), and undamaged mitochondria (mt). It is noteworthy that WEHI-164 cells typically exhibit a minimal number of intracellular vesicles. In comparison, L6 (C and D) cells display a less electron-dense cytoplasm with a predominantly euchromatic nucleus. Additionally, they often contain numerous lipid droplets (LD) and intracellular vesicles (IV), including phagolysosomes and autophagolysosomes with visible lamellar cellular debris.

Figure 9 demonstrates the impact of natural drug exposure on cancer cell morphology. CAPE did not significantly affected cell structure. The general morphology of the cells remains unaltered, and the mitochondria are mostly unaffected. However, there is an slightly increase in the number of intracellular vesicles (IV). Administration of CurE results (Figures 9C, D) in more pronounced alterations. The images demonstrate the presence of numerous intracellular vesicles, a blebbing plasma membrane with intact organelles inside blebs. The presented images suggest the occurrence of early (C) and late (D) apoptotic events, as evidenced by the presence of damaged apoptotic bodies (AB). The exposure of WEHI-164 cells to berberine (Figures 9E, F) resulted in an increased number of visibly damaged mitochondria (mt) and visible distor