Impact of PAC, a curcumin analog on the radiosensitivity of breast cancer cells

1. Introduction

Natural products have long played a central role in traditional medicine for centuries, treating a wide range of human ailments (Safarzadeh et al., 2014). Curcumin is derived from turmeric, a botanical substance that has received extensive scientific attention. Historically, turmeric has been incorporated into Indian and Chinese medicinal practices for its wide range of therapeutic effects (Fuloria et al., 2022; Oglah et al., 2020). Curcumin exhibits multiple pharmacological properties, indicating its potential for the treatment of chronic diseases and cancer prevention (Cozmin et al., 2024; Dipalma et al., 2024; Gupta et al., 2013). Extensive research has evaluated curcumin as a chemo-preventive compound in various cancer systems, supported by its antioxidant, anti-inflammatory, and anticancer effects (Purgatorio et al., 2024). When combined with standard cancer treatments such as chemotherapy or radiotherapy, curcumin can enhance therapeutic outcomes and reduce adverse side effects, broadening its clinical potential (Veselá et al., 2024). Much of curcumin’s biological activity is attributed to its inhibition of nuclear factor kappa-light-13 chain-enhancer of activated B cells (NF-κB), an inflammation key regulator and survival signaling pathways (Taniguchi & Karin, 2018). Aberrant inducement of NF-κB contributes to the pathogenesis of various diseases, including inflammatory disorders and cancer (Singh et al., 2015). NF-κB regulates a broad set of genes involved in sustaining growth of cells, besides preventing programmed cell death. It signaling network intersects with several major cellular pathways, including signal transducer and activator of transcription 3 (STAT3), activator protein-1 (AP-1), hypoxia-inducible factor-1 (HIF-1), interferon regulatory factors (IRFs), notch, nuclear factor erythroid 2-related factor 2 (NRF2), and the canonical Wnt signaling pathway (WNT/β-catenin) axis. Activation of NF-kB contributes to nearly every recognized hallmark of cancer. In the context of radiotherapy, NF-κB enhances cell survival by upregulating multiple anti-apoptotic genes, particularly the Bcl-2 family as well as inhibitor of apoptosis proteins (IAP) proteins, including cellular inhibitor of apoptosis protein 2 (c-IAP2). It also promotes resistance to radiation-induced damage by improving cellular antioxidant mechanisms. (Singh et al., 2015).

Most anticancer treatments induce cell death by triggering apoptosis in cancer cells (Peng et al., 2022). Efficient clearance of apoptotic cells by phagocytes, including dendritic cells and macrophages, is essential for maintaining immune tolerance and preventing chronic inflammation or autoimmune diseases (Cummings et al., 2016). In this context, NF-κB plays a critical regulatory role (Hoesel & Schmid, 2013; Munn, 2017). Among the signaling pathways regulated by NF-κB, TP53 is a key transcription factor that is often mutated or inactivated in many cancers. TP53 controls vital cellular processes, including cell cycle progression, senescence, and apoptosis (Alsbeih et al., 2007; Wang et al., 2023). It also strongly inhibits inflammation and tumor development, and affects cellular responses to chemotherapy and radiation therapy (Barabutis et al., 2018; Lindqvist et al., 2014; Marei et al., 2021; Tchelebi et al., 2014; Wei et al., 2023).

Curcumin offers significant health benefits as a result of its anti-proliferative, antioxidant properties, and anti-inflammatory, which arise from interactions with various molecular targets (Islam et al., 2024; Sivani et al., 2022; Yang et al., 2022). Nevertheless, the clinical application of curcumin is controlled by the low bioavailability, which results from its low solubility in water, limited oral absorption, low blood concentrations, short half-life, restricted tissue distribution, and rapid metabolism (Oglah et al., 2020; Sohn et al., 2021). Curcumin predominantly accumulates in the intestine, colon, and liver, potentially explaining its enhanced efficacy in the treatment of gastrointestinal disorders relative to other tissues (Dulbecco & Savarino, 2013).

To address curcumin’s low bioavailability, several approaches have been advanced to improve the absorption issue (Bertoncini-Silva et al., 2024; Hegde et al., 2023). One approach uses adjuvants such as piperine to modify curcumin’s metabolism and enhance bioavailability (Bisht & Maitra, 2009). Innovative delivery systems, including liposomes, nanoparticles, phospholipid complexes and micelles, aim to extend circulation time, improve tissue permeability, and increase resistance to metabolic degradation (Chauhan et al., 2014; Kaur et al., 2024). An alternative approach focuses on generating synthetic structural analogs that improve the pharmacological characteristics of natural dietary compounds. This approach is designed to address key challenges, including poor solubility, limited in vivo bioavailability, and suboptimal pharmacokinetic characteristics. (Mardaneh et al., 2025; Sarkar et al., 2010). Numerous curcumin derivatives have been developed with the goal of enhancing therapeutic potency while preserving their favorable safety profile (Moetlediwa et al., 2023).

NF-κB activation is widely involved in cancer-associated inflammation and undermines adaptive immunity, reducing the effectiveness of hormonal therapy, immunotherapy, radiation, and chemotherapy. However, inhibiting NF-κB alone has shown limited clinical benefit in cancer patients (Gilmore, 2021). NF-κB activation is widely involved in cancer-associated inflammation and undermines adaptive immunity, reducing the effectiveness of hormonal therapy, immunotherapy, radiation, and chemotherapy. However, inhibiting NF-κB alone has shown limited clinical benefit in cancer patients. The compound 3,5-Bis (4-hydroxy-3-methoxybenzylidene)-1-methylpiperidin-4-one (PAC), synthesized in our laboratory, represents a novel curcumin analog that exhibits enhanced stability in phosphate-buffered saline (PBS) and in circulation compared to curcumin (Al-Howail et al., 2016). PAC has shown increased potency and anticancer activity in various tumor models (Al-Howail et al., 2016; Al-Hujaily et al., 2011; Al-Qasem et al., 2016; Semlali et al., 2021). However, its effect in combination with radiotherapy has not been studied. The study hypothesizes that PAC, as a curcumin analog and NF-κB inhibitor, will enhance tumor cell destruction and overcome radiation resistance when used with radiotherapy. This effect is expected to result from suppression of the (NF-κB/Bcl-2) pathway in addition to activation of the (p53/Bax) pathway, thereby increasing radiosensitivity and improving anticancer treatment efficacy. Our research aims are to assess the cytotoxic effect of PAC in breast cancer (BC) cell lines and to determine its radiosensitizing properties when combined with X-ray irradiation. To achieve these aims, PAC’s effects on apoptosis, cell viability, and gene expression in irradiated breast cancer cells were evaluated

2. Result

2.1 Effect of PAC on cell proliferation

The real-time cell analysis system (RTCA) cell proliferation assay was used to assess PAC cytotoxicity in BC cell lines. RTCA measurements after PAC treatment were normalized by comparing the cell index (CI) values of treated wells to those of untreated controls (0 μM). Higher PAC doses resulted in a steady decrease in CI values in both cell lines as Fig. 1 demonstrates. The greatest reduction occurred at the 24-hour mark and continued throughout the 72-hour period. At 100 μM, CI was significantly reduced in both BC cells. In MCF-7 cells, CI (%) decreased to 38.4% (SE = 6.7) and 33.4% (SE = 6.9) at 72 and 96 hours, respectively. While, in MDA-MB-231 cells, CI (%) dropped to 20.8% (SE = 2.6) and 18.4% (SE = 1.2) at the same point.

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Fig. 1. Normalized cell index (CI%) effect of PAC on BC cell lines treated with various concentrations of PAC over 96 hours. Data points represent the average CI ± SE.

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2.2 Effect of PAC on clonogenic survival

PAC, a curcumin analog, inhibited colony formation in BC cell lines in a dose-dependent manner across concentrations 0.1 to 100 μM as shown in Fig. 2. The values of PAC IC50 in MCF-7 were 5.8 μM and, in MDA-MB-231 were 1.1 μM in MCF-7 cells consistent with previous studies (Cozmin et al., 2024; Semlali et al., 2021). For subsequent experiments, PAC was used at 5 μM and, 1 μM for MCF-7 and MDA-MB-231 cells.

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Fig. 2. Effect of PAC on BC cell line proliferation. Data represent the average of three independent experiments. Error bars represent the SE of the mean.

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2.3 Effect of PAC on apoptosis

Apoptotic response of BC cells to PAC treatment was assessed using flow cytometric analysis using Annexin V and propidium iodide (PI) staining. In Fig. 3(a), the cell distribution is shown across four categories: viable, early apoptotic (Annexin V+), late apoptotic (Annexin V+/PI+), and necrotic (PI+). The majority of cells (>80%) remained viable across all treatments; however, both BC cell lines exhibited an increased proportion of dead cells following PAC and/or irradiation compared to controls. Fig. 3(b) summarizes the combined percentages of dead cells for each treatment group. Both PAC and irradiation induced a dose-dependent increase in cell death, as demonstrated by t-test analysis, which confirmed statistical significance (p < 0.05). These findings suggest that apoptosis can be promoted by PAC in BC cells, as shown by increased phosphatidylserine exposure and compromised membrane integrity. DMSO, used as the PAC solvent, did not induce apoptosis at the tested concentration. As a positive control, exposure to 10 Gray radiation can increase apoptosis in both MCF-7 and MDA-MB-231 cells significantly, reaching 14.1% (SE = 2.3) and 17.5% (SE = 2.0), respectively. Combining PAC with radiation produced an additive enhancement of apoptotic activity. In MCF-7 cells, treatment with 5 µM PAC plus 4 Gray induced 12.02% apoptosis, whereas in MDA-MB-231 cells, PAC at 1 µM combined with 4 Gray resulted in 8.86% apoptosis. Overall, these combination treatments yielded apoptosis levels of 9.48% and 7.88%, respectively. These relatively modest increase in apoptosis suggests that other cell death mechanisms, such as autophagy or mitotic catastrophe, or a potent cytostatic effect, may be primarily responsible for the observed loss of clonogenic survival.

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Fig. 3. Flow cytometric analysis of apoptosis in MCF-7 and MDA-MB-231 cells. (a) Percentages of cells at diverse treatments. (b) Combined percentages of dead cells at multiple treatments. Data represent the mean ± SE of three independent experiments. Statistical significance was determined utilizing a t-test. Stars signify statistically significant difference (*p < 0.05) between groups.

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2.4 Effect of PAC on clonogenic cell survival curves

Evaluated whether PAC enhances the radiosensitivity of BC cells using clonogenic survival assays after a 4 hour incubation with PAC at its IC50 concentration. Fig. 4, showed that both cells exhibited exponential decreases in survival with increasing radiation doses. The surviving fraction at 2 Gy (SF2) was 0.26 [95% CI: 0.24–0.28] in MCF-7 cells, whereas MDA-MB-231 cells showed an SF2 of 0.48 [95% CI: 0.44–0.53], indicating nearly a twofold difference in radiosensitivity. The D50 values (irradiation required to reduce survival up to 50%) in the case of MDA-MB-231 were 1.81 Gy and 1.17 Gy for MCF-7. PAC pretreatment further enhanced radiosensitivity, lowering SF2 to 0.19 [95% CI: 0.17–0.21] in MCF-7 cells and to 0.38 [95% CI: 0.34–0.43] in MDA-MB-231 cells, corresponding to 25% and 21% increases in cell killing, respectively. Survival rates at each radiation dose, with and without PAC, were compared using a two-sample t-test. For MCF-7 cells, the values were: 1 Gy (0.04), 2 Gy (0.006), 4 Gy (0.09), and 6 Gy (0.05). In case of MDA-MB-231 cells, the values were: 1 Gy (0.01), 2 Gy (0.14), 4 Gy (0.30), and 6 Gy (0.09). Analysis of the full survival curves showed a statistically significant difference between combined treatment and radiation alone (One way repeated measures ANOVA: p = 0.004 for MCF-7, p = 0.002 for MDA-MB-231). These data show that combining PAC with irradiation heightens the radiosensitivity of both breast cancer cell lines. The results of the Chou-Talalay combination index (CI_C-T) analysis is given in Table 1. In both cell strains, lower doses were required to produce a median-effect dose (Dm), supporting that PAC potentiates the effect of IR. The CI_C-T values were less than one (CI_C-T < 1) in the combined treatment, confirming the potential effect, which becomes stronger at lower survival. This means that PAC substantially amplifies the impact of radiation, a synergy that is more pronounced at higher radiation doses.

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Fig. 4. Clonogenic survival curves of BC cells following X-ray irradiation with and without PAC (IC50 cytotoxicity). Data represents the average of three independent experiments. Error bars represent the SE of the mean.

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Table 1. Results of Chou-Talalay combination index (CI_C-T) analysis on cell survivals following ionizing radiation (IR) and the PAC combined treatment.

Cell strain:	MCF-7		MDA-MB-231
Treatment:	IR	IR+PAC	IR	IR+PAC
Dm*:	1.17	0.96	1.81	1.38
Combination index (CIC-T):	CIC-T	CIC-T	CIC-T	CIC-T
Fa** 0.75	0.75	0.62	1.14	0.82
Fa 0.90	0.48	0.40	0.72	0.48
Fa 0.95	0.36	0.29	0.53	0.34
Fa 0.97	0.29	0.24	0.42	0.26

*Dm: median-effect dose, which is the dose required to achieve 50% cell killing.

**Fa: fraction affected (Fa = 1 – Survival Fraction).

Notes: Lower dose needed with combined treatment to achieve the same level of effect indicates potentiation. A CIC-T of less than (<) one (1) indicates a synergistic effect of combined treatment. The lower the CIC-T, the stronger the synergism.

2.5 Effect of PAC on gene expression

The PAC impact on BC cells was investigated by conducting real-time PCR to quantify key molecular markers. A 24-hour incubation with 5 μM PAC was carried out for the treated cells. and subsequently irradiated after four hours and collected three hours after irradiation for gene expression analysis. In MCF-7 cells, PAC alone reduced NF-κB and Bcl-2 gene expression, which are associated with cell survival and apoptosis inhibition. PAC also increased expression of the pro-apoptotic genes p53 and Bax, especially when combined with radiation. Obviously, MDA-MB-231 cells exhibited greater resistance to radiation compared with MCF-7 cells, as reflected in their gene expression patterns. However, treatment with PAC alone reduced the NF-κB and Bcl-2 expression while elevating p53 and Bax levels in MDA-MB-231 cells (Fig. 5). That suggests the ability of PAC to promote apoptosis in BC cells by regulating genes associated with apoptosis and inflammation.

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Fig. 5. Effect of PAC on NF-κB, p53, Bcl-2, and Bax gene expression analyzed by RT-PCR in the BC cell line. Bars represent the mean ± SE of four independent experiments. Statistical significance was determined utilizing a t-test. Stars indicate statistical significant differences (*p &lt; 0.05) between groups.

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2.6 Effect of PAC on protein expression

To assess the effects of PAC on the expression of p53, Nf-κB, Bax, and Bcl-2 proteins West-ern blot analysis was conducted. Western blot analysis showed trends in protein level changes that were qualitatively consistent with the gene expression data, particularly for Bax in MDA-MB-231 cells and NF-κB in both lines, though these changes did not reach statistical significance (Fig. 6). A detailed discussion of these trends is presented in the Discussion.

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Fig. 6. Effect of PAC (5 μM) on protein expression analyzed by Western blot on the NF-κB, p53, Bcl-2, and Bax in the BC cell line. Bars represent the average protein expression. Error bars represent the SE from four independent experiments.

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3. Discussion

The activation of NF-κB in cancer-associated inflammation facilitates tumor growth, contributes to therapy resistance, and induces immune suppression, highlighting its significance as a therapeutic target. Sustained activation of NF-κB enhances resistance to radiation. In contrast, inhibition of NF-κB, such as with curcumin administration, may protect normal tissues and increase tumor sensitivity to radiotherapy by modulating multiple signaling and epigenetic pathways. This study validated the effectiveness of PAC in targeting BC cells. The anticancer properties of PAC were confirmed across several complementary experimental approaches. Real-time growth monitoring revealed that PAC affected proliferation and viability of both BC cell lines in a concentration- and time-dependent manner (Fig. 1). Higher PAC concentrations (50 and 100 μM) produced a marked decline in CI values, reducing them to less than 50% between 48 and 96 hours. In contrast, minimal changes were observed with lower doses (0.1–10 μM), where CI values remained near 70–80% throughout the same period. These results align with earlier reports on the curcumin analog B14, which was found to suppress the proliferation of MCF-7 and MDA-MB-231 cells, with IC₅₀ values of 8.84 μM and 8.33 μM, respectively.(Shen et al., 2021).

To further examine the chemotherapeutic potential of PAC, clonogenic assays were used to quantify long-term survival of breast cancer cells following exposure to both low and high PAC concentrations (Fig. 2). The IC₅₀ values derived from colony-forming assays were for MCF-7 cells 1.11 μM and for MDA-MB-231 cells 5.86 μM. These results parallel earlier reports for PAC (Al-Hujaily et al., 2011), and the curcumin analog B14 (Shen et al., 2021), which demonstrated substantial suppression of clonal growth at comparable micromolar doses relative to curcumin and DMSO controls.

Our results also align with prior reports that describe the potent pro-apoptotic activity of PAC in BC cells. Al-Howail et al., (2016) demonstrated that PAC triggers apoptosis in both MCF-7 and MDA-MB-231 cells at micromolar concentrations, which is in agreement with the present findings. Earlier studies have also demonstrated that PAC modulates critical signaling pathways involved in cancer cell survival as well as progression (Al-Mohanna et al., 2023; Cozmin et al., 2024). In this study, apoptosis assays further confirmed the pro-apoptotic activity of PAC (Fig. 3), with increased cell death observed in both the PAC-only and combined treatment groups. These results support previous reports, including those by Al-Howail et al., (2016), which showed potent apoptotic effects of PAC. While PAC alone caused a limited rise in late apoptosis, its combination with radiation produced a more substantial apoptotic response, reflected by reduced proliferation and clonogenicity and overall higher levels of cell death (Sak, 2020). Mechanistically, PAC-mediated inhibition of NF-κB may relieve suppression of p53 signaling through established regulatory cross-talk between these pathways, thereby enhancing downstream pro-apoptotic signaling involving Bax and Bcl-2, which may partially underlie the apoptotic and radiosensitizing effects observed following PAC treatment. Collectively, these findings suggest that PAC may enhance the therapeutic response to radiation in breast cancer and potentially improve clinical outcomes. Further investigation is needed to clarify the involvement of particular apoptotic mediators, such as Bcl-2 family proteins and caspases, in PAC- driven cell death. It is also important to note that the reduction in cell number shown in Fig. 1 represents total cytotoxicity. The relatively low apoptosis levels in Figs. 2 and 3(b) indicate that apoptosis may not be the primary mechanism of PAC-induced cell killing in these solid tumor models. Other death pathways may contribute to the observed cytotoxicity, such as mitotic catastrophe or senescence. Evaluating PAC in combination with additional chemotherapeutic or targeted agents may further strengthen its therapeutic potential.

Clonogenic survival analysis was used to assess how PAC influences the radiation response of breast cancer cells. As illustrated in Fig. 4, increasing radiation doses produced a pronounced exponential reduction in cell survival. PAC treatment significantly reduced cell survival following radiation exposure in both breast cancer cell lines. One-way repeated measures ANOVA indicated a significant difference between the combined treatment group and the radiation-only group (p = 0.004 for MCF-7; p = 0.002 for MDA-MB-231). The surviving fraction at 2 Gy (SF2) decreased from 0.26 to 0.19 in MCF-7 cells and from 0.48 to 0.38 in MDA-MB-231 cells after PAC exposure, corresponding to reductions of 25% and 21%, respectively. These results demonstrate that PAC markedly increases radio sensitivity and enhances the cytotoxic effects of radiation. The Chou-Talalay combination index (CI_C-T) analysis indicated synergism (CI_C-T < 1) that becomes stronger as the radiation dose increases (Table 1). This is a very positive finding, indicating that the drug significantly enhances the effect of radiation, especially at doses that achieve high cell killing. This suggests that the combination treatment is more effective at reducing cancer cell survival than radiation alone, which could indicate a synergistic effect (Zoi et al., 2021). These results provide additional evidence that PAC can significantly improve the effectiveness of radiation therapy by reducing cell viability (M. M. Islam et al., 2024). Taking together, these study outcomes highlight the essential part of PAC in enhancing the radiation treatment efficacy in BC cell lines. This emphasizes the therapeutic promise of combining PAC with radiotherapy and highlights the necessity for further investigation into its mechanisms of action and potential clinical applications.

Gene expression studies consistently demonstrate that curcumin is pharmacologically safe and offers significant health benefits, particularly in cancer, due to its anti-inflammatory and antioxidant properties. These effects are likely mediated by suppression of NF-κB and other mechanisms (M. R. Islam et al., 2024). While curcumin inhibits NF-κB signaling, many chemotherapeutic agents upregulate genes linked to cell proliferation, invasion, survival, and metastasis (Aggarwal et al., 2005).

A wide range of curcumin derivatives has been explored for their anti-inflammatory and antiproliferative properties.(Kaur et al., 2024). Several heterocyclic cyclohexanone-based analogs of curcumin have also been synthesized (Yadav et al., 2010) and their ability was examined to suppress NF-κB transactivation (Katsori et al., 2015). Among these compounds, PAC has been shown to enhance the expression of DNA repair–related genes in BC cell lines (Almalki et al., 2023). Furthermore, PAC reduces cellular proliferation, prompts both autophagy and apoptosis, and mitigates oxidative stress. (Semlali et al., 2021). PAC modulates several signaling pathways, such as NF-κB, Wnt, and MAPK (Semlali et al., 2023). These results corroborate previous studies indicating that curcumin inhibits breast cancer cell proliferation and regulates genes associated with apoptosis, angiogenesis, cell cycle control, and metastasis (Nagaraju et al., 2012).

This study shows that PAC reduces NF-κB expression in both BC cells (Fig. 5), consistent with previous research. In MCF-7 cells, combining PAC with radiation further increases pro-apoptotic gene expression (p53 and Bax) compared to PAC alone. PAC alone also reduces Bcl-2 expression relative to the control. PAC lowers NF-κB expression, with an even greater reduction observed when paired with radiation. Within MDA-MB-231 cells, the combined PAC–radiation treatment produced a more pronounced reduction in NF-κB expression compared with either PAC alone or the control group. This treatment also caused a slight elevation in p53 levels. Exposure to PAC alone resulted in reduced Bcl-2 expression while elevating Bax protein levels, further supporting its pro-apoptotic activity in MDA-MB-231 cells. The statistically significant alterations in gene expression (p < 0.05) indicate that PAC promotes apoptosis in BC cells by influencing key inflammatory and apoptotic regulatory pathways. The data also indicate that combined treatment exerts distinct effects on gene regulation, reflecting potential biological and therapeutic differences between the two cell lines (Fig. 5). A comparable investigation (Quispe-Soto & Calaf, 2016) The authors reported that, in MDA-MB-231 cells, exposure to 30 μM curcumin led to elevated levels of p53 and Bax, whereas Bcl-xL expression was reduced compared with the negative control. In contrast, treatment of MCF-7 cells with 15 μM curcumin resulted in lowered expression of NF-κB, p53, and Bcl-xL, while Bax was increased. It is worth noting that the curcumin concentrations used in that earlier study were substantially higher than those used in the present investigation. However, mRNA expression alone does not fully reflect the activity of post-translationally regulated proteins such as NF-κB, p53, and Bcl-2. In this study, the transcriptional changes were supported by Western blot analysis, confirming consistent effects at the protein level.

Western blot analysis provided additional insight into molecular alterations in key signaling proteins following exposure to PAC and radiation. The contrasting p53 status of the two cell lines—wild-type p53 in MCF-7 and mutant p53 (codon 280) in MDA-MB-231—is crucial for understanding their differential responses to treatment. In MCF-7 cells, the observed increases in Bax and p53 expression align with activation of the classical p53-dependent apoptotic pathway (Balcer-Kubiczek et al., 1995). Conversely, in MDA-MB-231 cells, the p53 mutation likely contributes to the different p53 and Bax dynamics. Mutant p53 often loses its tumor suppressor functions and can even gain oncogenic properties (Hui et al., 2006). Therefore, the observed increase in Bax expression induced by PAC in MDA-MB-231 cells, despite only a slight elevation in mutant p53, suggests the involvement of a p53-independent mechanism for Bax induction. Alternatively, PAC may mitigate the pro-survival effects commonly attributed to mutant p53. The observed decrease in NF-kB in MDA-MB-231 cells is also particularly notable in this context, as NF-kB is a crucial transcription factor known to promote inflammation, cell proliferation, and survival. This trend qualitatively suggests a potential anti-inflammatory and anti-proliferative effect of PAC, which could be pertinent for aggressive subtypes like MDA-MB-231, where inflammatory pathways are often hyperactive.

The protein expression observations show some qualitative alignment with previous research on PAC’s cellular effects in other cancer models. For instance, two studies in oral cancer cell models (Ca9-22) corroborate our observations, reporting that PAC treatment lowered protein levels of Bcl-2 and NF-kB and raised levels of Bax and p53, as measured by Western blot (Semlali et al., 2021). This visually consistent pattern across different cancer types suggests a potential general influence of PAC on these pathways. While some previous studies in BC models present variations (Almalki et al., 2023) These discrepancies likely stem from differences in experimental conditions, PAC source, or the inherent variations between gene expression and protein level regulation. The qualitative consistency of our protein-level observations with the oral cancer studies regarding p53, Bax, Bcl-2, and NF-kB, combined with partial agreement with breast cancer gene expression data for Bax and Bcl-2, underscores the apparent impact of PAC on these crucial pathways. In this context, the observed differences between the BC cell lines used in the present study further support the notion that PAC-induced responses may vary according to intrinsic cellular characteristics, reflecting cell line–specific biological behavior.

Additional research is required to elucidate how the anti-inflammatory and immunomodulatory actions of PAC contribute to its anticancer effects. Clarifying these mechanisms may facilitate the development of novel therapeutic strategies that target critical aspects of cancer. Limitations of these studies are inherent to challenges associated with curcumin analogs and in vitro testing. While these analogs aim to enhance curcumin’s bioavailability, achieving optimal delivery and distribution within complex vivo systems remain a significant hurdle. Furthermore, although specificity has been enhanced, the possibility of off-target effects and interactions with concomitant medications remains. Moreover, the results obtained from in vitro BC cell lines may not fully reflect the complex tumor microenvironment found in vivo, limiting direct clinical translation. Moreover, the heterogeneity of BC indicates that responses to the studied analog may differ substantially across various subtypes, underscoring the necessity for additional study.

4. Materials and Methods

5. Conclusions

PAC significantly promoted apoptosis and inhibited cell proliferation, both independently and when combined with radiation therapy. A marked decrease in cell survival (p < 0.01) and an increase in BC cell radiosensitivity were observed following PAC treatment, indicating a synergistic effect with radiation. Alterations in apoptotic markers, as well as the expression levels of p53, Bcl-2, and Bax, further support the potential of PAC as an effective anticancer agent. These findings underscore the promise of PAC as an adjunct to radiotherapy and support its continued development as a radiosensitizer for BC treatment.