Erica spiculifolia Extract Potentiates Cisplatin Cytotoxicity by Reactivating p53 and Caspase-3-Dependent Apoptosis in Colorectal Carcinoma

TL;DR

Erica spiculifolia extract enhances cisplatin's effectiveness in colorectal cancer by reactivating p53 and promoting apoptosis.

Contribution

The study demonstrates a synergistic effect of Erica spiculifolia extract with cisplatin through p53 reactivation and apoptosis induction in colorectal carcinoma cells.

Findings

Combination of ESE and cisplatin showed strong superadditive effects with Combination Index values below 1.

ESE treatment restored p53 and increased apoptotic cell death in HT-29 cells.

Kaempferol 3-O-glucoside, myricitrin, and naringenin 7-O-glucoside were identified as key bioactive compounds in ESE.

Abstract

Resistance to apoptosis represents a major limitation of platinum-based chemotherapy in colorectal carcinoma, frequently arising from impaired p53 signaling and inefficient execution of programmed cell death. In this study, we investigated the anticancer activity of Erica spiculifolia extract (ESE) and its ability to synergistically enhance cisplatin cytotoxicity in HT-29 colorectal carcinoma cells. Cell viability was assessed using the MTT assay, followed by formal combination analysis based on the Chou–Talalay methodology. Combination experiments employed a non-constant ratio regimen in which a fixed ESE concentration (45 µg/mL) was combined with serial cisplatin dilutions (45.0–2.8 µg/mL) to define interaction behavior across multiple effect levels. Quantitative analysis revealed a strong superadditive effect, with Combination Index values well below 1 and markedly elevated Dose Reduction Indices for cisplatin, indicating substantial dose-sparing across effect levels. To elucidate the molecular basis of this synergism, apoptosis-related protein expression was profiled using a membrane-based immunoassay. Combined ESE and cisplatin treatment induced full-scale p53 reactivation, including restoration of phosphorylated p53 isoforms associated with DNA damage-dependent apoptotic signaling. Acridine orange/propidium iodide staining confirmed a pronounced increase in early and late apoptotic/necrotic cells following combination treatment. UHPLC-HRMS analysis identified kaempferol 3-O-glucoside (8830.19 ± 11.01 ng/mg dw) and myricitrin (3074 ± 3.12 ng/mg) as predominant flavonols, followed by naringenin 7-O-glucoside (5958.96 ± 9.98 ng/mg), while chlorogenic, cinnamic, quinic, and gallic acids were the main phenolic acids detected. These constituents may contribute to HT-29 cell sensitization to cisplatin.

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1. Introduction

Colorectal carcinoma (CRC) is defined not only by its high incidence but also by its pronounced capacity to tolerate cytotoxic stress, a feature that underlies treatment failure in advanced disease. According to the Global Cancer Observatory, CRC remains among the most commonly diagnosed malignancies worldwide and a leading cause of cancer-related mortality, with projections indicating a continued increase in global burden over the coming decades [1,2]. Despite advances in surgical resection, adjuvant chemotherapy, and molecular stratification, durable disease control in advanced CRC remains difficult to achieve, largely due to adaptive resistance mechanisms rather than insufficient initial tumor response [3,4].

A defining limitation of cytotoxic chemotherapy in CRC is the uncoupling of DNA damage from effective apoptotic execution. Cisplatin is a prototypical DNA-damaging agent whose anticancer activity relies on the formation of intra- and interstrand DNA crosslinks that activate stress response signaling and programmed cell death [5]. However, CRC cells frequently display reduced sensitivity to cisplatin compared with other solid tumors, a phenomenon attributed less to impaired DNA damage induction and more to defects in downstream apoptotic signaling [6]. Consistent with the MOC-5 paradigm of apoptosis-related chemoresistance in colorectal cancer, resistance is often driven by alterations in apoptosis regulation, including impaired activation of p53-dependent pathways and downstream executioner caspases [7]. These observations suggest that defective apoptotic commitment represents a critical determinant of cisplatin resistance in CRC and highlight restoration of apoptosis as a rational strategy for chemosensitization.

The tumor suppressor p53 occupies a pivotal position in coordinating cellular responses to genotoxic stress. Upon activation, p53 regulates transcriptional programs that determine cell fate, promoting either cell cycle arrest or apoptosis depending on damage severity. In CRC, p53 signaling is frequently compromised not only by TP53 mutations but also by dysregulation of post-translational modifications that attenuate its pro-apoptotic output [6,8,9]. Experimental evidence indicates that deficient or defective p53 activation contributes directly to cisplatin resistance by allowing CRC cells to survive extensive DNA damage without engaging cell death pathways [10]. Approximately 50% of human colorectal carcinomas harbor TP53 mutations that result in dysfunctional p53 expression, a defect that has been strongly associated with limited responsiveness to anticancer treatment, including newer generations of platinum-based drugs such as oxaliplatin [11].

Combination therapy represents a rational strategy for overcoming apoptosis resistance by targeting complementary nodes within the cell death machinery. Rather than escalating cytotoxic drug doses, synergistic combinations can lower the apoptotic threshold, allowing effective tumor cell elimination at reduced exposure levels. Validated quantitative frameworks such as the Chou–Talalay method enable rigorous and predictive assessment of synergistic interactions, providing a rational basis for the design and interpretation of combination treatment strategies [12].

Natural products and phytochemical-rich extracts have re-emerged as promising candidates for chemosensitization because of their ability to modulate multiple stress- and apoptosis-related pathways simultaneously. Numerous plant-derived compounds have been shown to enhance p53 activity, promote caspase-3 activation, and sensitize cancer cells to platinum-based chemotherapy while exhibiting relatively low toxicity toward non-malignant tissues [13]. A growing body of in vitro and in vivo evidence indicates that phytochemicals can interfere with cancer initiation and progression both independently and in a synergistic manner by modulating key signaling networks in cell survival, stress responses, and apoptosis [14]. In colorectal cancer models, flavonoid and non-flavonoid metabolites have been reported to lower the threshold for apoptosis induction and restore chemotherapy responsiveness through coordinated modulation of pro-death signaling [15,16].

Within this context, species of the Ericaceae family have attracted increasing interest due to their distinctive phytochemical composition and biological activity. Erica spiculifolia Salisb., an evergreen shrub native to the Balkan region, has traditionally been used for its antioxidant and anti-inflammatory properties [17,18]. Recent phytochemical profiling has revealed that E. spiculifolia extracts are rich in proanthocyanins, phenolic acids, flavonoids, and triterpenoid compounds—metabolite classes frequently implicated in regulation of oxidative stress responses and apoptosis [19]. Importantly, contemporary in vitro studies have demonstrated that E. spiculifolia extracts exert cytotoxic effects against a panel of human cancer cell lines, with favorable selectivity toward malignant cells [20]. Despite these promising observations, the molecular basis of these activities remains incompletely defined, as does the capacity of E. spiculifolia extracts and their constituents to potentiate chemotherapy-induced apoptosis.

In the present study, we investigate the anticancer activity of Erica spiculifolia extract (ESE) in HT-29 colorectal carcinoma cells and examine its ability to potentiate cisplatin cytotoxicity. We integrate cytotoxicity profiling in different treatment regimens with formal synergy analysis using the Chou–Talalay methodology to define the nature and strength of the ESE-cisplatin interaction at varying exposure levels. In parallel, we employ proteome-based analysis of apoptosis-related proteins to elucidate molecular mechanisms underlying the observed synergistic effects, with a specific focus on reactivation of p53 signaling and caspase-3-dependent apoptosis. The multifaceted approach of our study provides first mechanistic insights into the chemosensitizing potential of E. spiculifolia, supporting its further exploration as a natural adjunct to platinum-based therapy in colorectal carcinoma.

2. Results and Discussion

2.1. UHPLC-HRMS Quantitative Analysis of Phenolic Compounds in E. spiculifolia

E. spiculifiolia was analyzed for phenolic constituents by UHPLC-HRMS (Table 1). Flavonoids were the main class, reaching 69.4% of the determined analytes. Kaempferol 3-O-glucoside (astragalin) and myricitrin were the dominant flavonols, being present at 8830 ng/mg and 3074 ng/mg dw, respectively, followed by the flavanone naringenin 7-O-glucoside (5958.96 ng/mg) (Table 1).

The aglycones were found in significantly lower concentrations (up to 29. 17 ng/mg quercetin). The extract was characterized by a high content of quinic acid and its conjugate chlorogenic acid. The quantity of analysed quinic and caffeoylquinic acid achieved 17.83% of the determined compounds. On the other hand, hydroxybenzoic acids accounted for 6.59%; their profile was delineated by gallic and gentisic acids. Cinnamic acid, alongside hydroxycinnamic derivatives, was also determined; the highest amounts of cinnamic acid (1119 ng/mg) should be distinguished. The plant extract displayed moderate content of flavanols (459 ng/mg).

2.2. Cytotoxicity Screening Results

The cytotoxic potential of Erica spiculifolia extract (ESE), the reference drug cisplatin, and their combination was evaluated in HT-29 colorectal carcinoma cells using the MTT viability assay. Dose-response analyses were performed to determine the half-maximal inhibitory concentrations (IC_50_) for each treatment regimen, allowing a comparative assessment of their antiproliferative efficacy. The resulting IC_50_ values, expressed as mean ± standard deviation, are summarized in Table 2.

ESE alone exhibited moderate cytotoxic activity against HT-29 cells, with an IC_50_ value of 40.3 µg/mL, whereas cisplatin monotherapy showed an IC_50_ of 33.8 µg/mL, corresponding to 112.7 µM. As illustrated in the bar-graph representation of cell viability (Figure 1), co-treatment with a fixed concentration of ESE consistently enhanced the cytotoxic effect of cisplatin across all tested exposure levels. This effect was associated with statistically significant reductions in cell viability compared with cisplatin monotreatment at corresponding concentrations (p < 0.05). The magnitude of the interaction resulted in an approximately 2- to 5-fold decrease in cell viability, with the most pronounced differences observed at lower cisplatin concentrations. Under these conditions, combination treatment achieved levels of growth inhibition exceeding those obtained with higher cisplatin doses alone. Accordingly, the IC_50_ of cisplatin in the combination regimen was reduced by nearly 15-fold, decreasing from 33.8 µg/mL (112.7 µM) to 2.3 µg/mL (7.7 µM). This pronounced shift indicates a strong sensitizing effect of ESE and supports its role as a potent adjuvant capable of markedly enhancing cisplatin efficacy in HT-29 colorectal carcinoma cells.

2.3. Results from the Combination Study

The synergistic interaction between Erica spiculifolia extract (ESE) and cisplatin was quantitatively evaluated using the Chou–Talalay method to characterize the magnitude and consistency of their combined effects on HT-29 colorectal carcinoma cells. This approach enabled a robust and quantitative assessment of drug interactions across multiple effect levels, based on experimentally derived dose-response relationships for each agent alone and in combination (Figure 2). Key interaction parameters, including the Combination Index (CI), Dose Reduction Index (DRI) (Table 3, Figure 3), and normalized isobologram analysis (Table 4, Figure 4) provide a comprehensive analytical framework for interpreting the extent of synergism, additivity, or antagonism between varying ratios of ESE and cisplatin and for identifying dose ranges in which the combination produces the greatest synergistic advantage.

An initial examination of the dose-response relationships (Figure 2) reveals a clear distinction between the combination regimen and the respective individual treatments, strongly suggestive of synergistic interaction. The curve corresponding to the combined treatment with Erica spiculifolia extract (ESE) and cisplatin (green) is characterized by both a pronounced leftward shift and a noticeably steeper slope compared with the curves obtained for ESE (blue) and cisplatin (red) alone. This pattern indicates that lower doses of the combined regimen are sufficient to achieve equivalent or greater fractions affected (Fa), while the sharper slope reflects a more efficient transition from sub-effective to highly cytotoxic concentrations. In contrast, the individual exposure to both ESE and cisplatin yields a more gradual increase in growth inhibition, which remains submaximal across the tested concentration range. Cisplatin alone elicits a stronger response than ESE but still requires relatively higher doses to achieve Fa values exceeding 0.5 (50% reduction in cell viability). Notably, the combination treatment consistently outperforms both single agents, yielding higher Fa values at corresponding doses. This trend is observed across the entire concentration range and is most evident at intermediate and higher effect levels, where the combination approaches near-maximal effect.

Table 3 provides complementary insight into the dose-effect relationship, the interaction metrics and the dose-sparing potential of the studied combination at each exposure level (actual treatment concentrations).

Central to the analysis is the Fa (fraction affected), representing the biological effect at each combination point as the fraction of growth inhibition relative to untreated controls. In the present dataset, Fa values span a relatively high range (0.59–0.93), indicating that the combination was evaluated primarily under conditions of moderate to strong cytotoxicity. This is particularly relevant for assessing synergy, as drug interactions often vary with effect level, and clinically meaningful synergy is most relevant at higher levels of tumor growth inhibition. In this non-constant ratio design, ESE was maintained at a fixed concentration (45 µg/mL), while cisplatin was serially diluted. This experimental configuration allows direct evaluation of whether ESE can sensitize cells to decreasing cisplatin doses, a key consideration in dose-reduction strategies aimed at minimizing cisplatin-associated toxicity. The observation that high Fa values are retained even at substantially lower cisplatin concentrations immediately suggests a superadditive contribution of the plant extract.

The Compusyn^Ⓡ^ software additionally provided predictive doses of ESE and cisplatin, required to achieve the same Fa when used in a monotreatment regimen. These theoretical single-agent doses thus serve as reference points for the interaction analysis and form the quantitative basis for the subsequent CI and DRI calculations. Across the dose-response datasets for both ESE and cisplatin, the estimated dose equivalents increase progressively with rising Fa, indicating that substantially higher doses would be required to achieve strong cytotoxic effects under single-agent conditions. For example, at Fa ≈ 0.87 (corresponding to the point of strongest synergism) the predicted single-agent ESE dose exceeds 300 µg/mL, and the equivalent cisplatin dose approaches 160 µg/mL; in contrast, only 45 µg/mL ESE and 11.2 µg/mL cisplatin were required to produce the same effect in the combined treatment regimen.

The Dose Reduction Index (DRI) analysis further substantiates the synergistic interaction between ESE and cisplatin by quantifying the fold reduction in the effective dose of each agent achieved through combination treatment. As DRI values greater than 1 indicate a favorable dose-sparing effect, the consistently elevated DRI values observed across all Fa levels provide strong quantitative support for enhanced combination efficacy (Table 3, Figure 3). Notably, the dose-reducing effect is particularly pronounced for the chemotherapy drug cisplatin, with DRI values ranging between 2 and 24. At Fa ≈ 0.59, the estimated DRI for cisplatin is approximately 2.0, indicating a twofold reduction in the drug dose required to achieve the observed effect. This dose-reduction effect becomes significantly more pronounced at higher Fa levels, reaching DRI values of approximately 13.6 at Fa ≈ 0.73 and 14.2 at Fa ≈ 0.87. Interestingly, the strongest boosting effect on cisplatin efficacy is observed at intermediate effect levels, peaking at Fa ≈ 0.70 (DRI = 24.1). However, even at the highest Fa examined (Fa ≈ 0.93), cisplatin retains a DRI of 6.19, demonstrating a strong and persistent synergistic effect across a broad cytotoxic range.

DRI values for ESE similarly exceed 1 across all Fa levels, although to a lesser extent than those observed for cisplatin. ESE DRI values increase from approximately 1.32 at Fa ≈ 0.59 to 2.59 at Fa ≈ 0.73, rise further to 6.80 at Fa ≈ 0.87, and reach a maximum of 14.14 at Fa ≈ 0.93. The comparatively more modest dose-reduction effect observed for ESE suggests that, within the combination regimen, its primary contribution is chemosensitization and modulation of cellular response pathways rather than acting as the principal cytotoxic component.

The Combination Index (CI) is the central quantitative descriptor of drug interaction. In the present dataset, four of the five combination points yield CI values substantially below 1, ranging from 0.22 to 0.49, consistent with moderate to very strong synergism (Table 3, Figure 3). The strongest synergy is observed at high fractional effects (Fa ≈ 0.87–0.93), where CI values fall below 0.25, indicating that the combined treatment is more than fourfold more effective than expected from simple additivity. One combination point at Fa ≈ 0.59 exhibits a CI slightly above 1, indicative of near-additive or mildly antagonistic behavior. Importantly, this single data point occurs at a lower Fa and does not detract from the overall synergistic profile observed across higher, therapeutically more relevant effect levels.

The normalized dose ratios (Table 4) quantify the fractional contribution of each agent to the combined effect relative to its predicted single-agent requirement. Values well below unity (<1) indicate that the observed cytotoxicity is achieved using only a fraction of the doses expected under single-agent conditions. In the present analysis, normalized ratios for both agents are consistently reduced across all data points, with particularly low values observed for cisplatin, reflecting a pronounced dose-sparing effect. Under mutually nonexclusive assumptions, the sum of these normalized ratios corresponds directly to the CI, thereby providing a quantitative link between the tabulated interaction parameters and the normalized isobologram (Figure 4).

Consistent with this numerical analysis, the plotted points in the isobologram are positioned predominantly below the line of additivity, demonstrating that the combination of ESE and cisplatin achieves equivalent cytotoxic effects at substantially reduced fractional doses of both agents. The strong reduction in normalized cisplatin ratios, especially at higher fractional effects, is of particular relevance given the dose-limiting toxicity of platinum-based chemotherapy and underscores the chemosensitizing capacity of ESE.

2.4. Proteome Profiling Results

To gain mechanistic insight into the observed cytotoxic and synergistic effects of ESE and cisplatin, changes in the expression of key apoptosis-related proteins were examined using a membrane-based proteome profiling approach. HT-29 colorectal carcinoma cells were exposed to equi-inhibitory concentrations of Erica spiculifolia extract (ESE), cisplatin, or their combination, and the resulting protein expression patterns were compared with untreated control cells (Figure 5).

Proteome profiler analysis revealed pronounced and pathway-specific alterations in apoptosis- and stress-related proteins across all treatment groups, providing mechanistic insight into the differential cellular responses to Erica spiculifolia extract (ESE), cisplatin, and their combination in HT-29 colorectal carcinoma cells (Figure 5 and Figure 6). Importantly, the data highlight a clear divergence between apoptotic execution and upstream regulatory control, which appears to underlie both chemoresistance to cisplatin in the monotreated group and the strong synergistic response observed under combination treatment.

A dominant and consistent finding across all treated groups was the maximal activation of cleaved caspase-3, which reached 100% of the maximal detectable signal in the ESE, cisplatin, and ESE + cisplatin groups, compared with only 9% basal expression in untreated cells. Caspase-3 is the principal executioner caspase and represents a convergence point for both intrinsic and extrinsic apoptotic pathways. Its robust activation indicates that, regardless of upstream signaling differences, all treatments ultimately engage the canonical execution phase of apoptosis. However, our data also underscore that executioner caspase activation per se does not guarantee irreversible cell death or durable cytotoxicity. In malignant cells, caspase activation can be transient or incomplete, allowing subsets of cells to recover from near-death states via apoptotic escape or anastasis when upstream stress-response signaling remains defective. This phenomenon is particularly relevant in colorectal carcinoma, where transient caspase activation may fail to induce irreversible cell death when p53 signaling is compromised. Cancer cells can undergo anastasis, recovering from the brink of apoptosis despite DNA damage, in part through MDM2-mediated degradation of p53, which enables cell survival. Within this context, the divergence between similar levels of cleaved caspase-3 and markedly different long-term cytotoxic outcomes among the treatment groups suggests that ESE and cisplatin monotreatments predominantly induce a form of “abortive” or reversible apoptosis, whereas the combination regimen enforces a fully committed apoptotic program that precludes cell survival [21,22].

Indeed, analysis of p53 phosphorylation states revealed striking treatment-dependent differences that provide a mechanistic explanation for both the limited efficacy of cisplatin monotherapy and the pronounced synergism observed with ESE co-treatment. In the ESE-treated group, all analyzed p53 phospho-isoforms were markedly reduced compared with untreated controls, with p53 Ser15 decreasing to 26.6%, p53 Ser46 to 36.4%, and p53 Ser392 to 19.2% of maximal expression. This pattern suggests that, while ESE is capable of activating caspase-3, it simultaneously suppresses upstream p53 stress signaling. Such a profile is consistent with phytoprotective or antioxidant effects of plant extracts, rather than full pro-apoptotic commitment. Numerous plant-derived phytoconstituents exhibit similar dual behavior, acting as cytoprotective agents under moderate stress by attenuating DNA damage signaling and preserving cellular integrity. Accordingly, ESE alone may favor a scenario in which caspase-3 is transiently activated but cells retain sufficient stress-response plasticity to undergo apoptotic reversal and sustain survival.

Cisplatin monotreatment elicited a distinct yet suboptimal p53 response in HT-29 cells. Although phosphorylation at Ser46 (80.4%) and Ser392 (30.0%) was partially preserved, Ser15 phosphorylation remained markedly suppressed (29.0%). Functionally, phosphorylation at Ser15 represents an early DNA damage–responsive modification that promotes p53 stabilization by limiting MDM2-mediated degradation. In contrast, Ser46 phosphorylation is preferentially associated with transcriptional activation of pro-apoptotic target genes, while Ser392 enhances p53 tetramerization and DNA-binding affinity, thereby reinforcing transcriptional competence. The incomplete activation pattern observed under cisplatin monotreatment therefore suggests a functional decoupling of DNA damage sensing from full pro-apoptotic transcriptional execution—a phenomenon frequently associated with cisplatin-resistant colorectal carcinoma cells [11]. Such partial p53 activation may account for the presence of caspase-3 activation without sustained tumor cell elimination, indicating that apoptotic signaling is initiated but not irreversibly enforced.

In sharp contrast, the combined ESE and cisplatin treatment restored coordinated, full-scale p53 activation, with Ser15 phosphorylation reaching 91.2%, and Ser46 and Ser392 both reaching 100% of maximal signal. The simultaneous activation of all three sites therefore represents a fully competent tumor suppressor response, providing a plausible molecular basis for the observed synergism and for overcoming cisplatin resistance in the HT-29 model [8]. Our findings suggest that ESE potentiates cisplatin efficacy by restoring coordinated, site-specific p53 activation, thereby converting a partial and potentially reversible stress response into a stabilized, apoptosis-committed state. Under these conditions, caspase-3 activation operates within a stabilized state of a pan-p53 activation that drives irreversible apoptotic commitment, leaving limited capacity for anastatic recovery.

Cisplatin monotreatment exhibited a distinct but still suboptimal p53 response. While p53 Ser46 and Ser392 phosphorylation levels were partially preserved (80.4% and 30.0%, respectively), p53 Ser15 remained suppressed (29.0%). Phosphorylation at Ser15 is a critical early event in DNA damage recognition and p53 stabilization, while Ser46 phosphorylation is more directly associated with apoptotic gene transcription. The incomplete activation of these sites suggests a decoupling of DNA damage sensing from proapoptotic transcriptional responses, a phenomenon frequently observed in cisplatin-resistant colorectal carcinoma cells [11]. Such partial p53 activation may explain why caspase-3 is activated but fails to translate into sustained tumor cell elimination.

In sharp contrast, the ESE + cisplatin combination restored full-scale p53 activation, with p53 Ser15 reaching 91.2%, Ser46 reaching 100%, and Ser392 also reaching 100% of maximal signal. This comprehensive activation of p53 phosphorylation sites strongly indicates reestablishment of a functional DNA damage response and apoptotic transcriptional program. Phosphorylation at Ser15 stabilizes p53 and prevents MDM2-mediated degradation, while Ser46 is specifically linked to transcription activation of pro-apoptotic genes. Ser392 enhances p53 tetramerization and DNA binding affinity. The simultaneous activation of all three sites therefore represents a fully competent tumor suppressor response, providing a plausible molecular basis for the observed synergism and for overcoming cisplatin resistance in the HT-29 model [8]. Under these conditions, caspase-3 activation operates within a stabilized state of a pan-p53 activation that drives irreversible apoptotic commitment, leaving limited capacity for anastatic recovery.

Further insight into mitochondrial apoptotic regulation was obtained by examining Smac/DIABLO expression. Smac/DIABLO antagonizes inhibitor of apoptosis proteins (IAPs) and facilitates caspase activation following its release from mitochondria. Interestingly, steady-state Smac/DIABLO levels were nearly halved in the ESE group (46.2%) and the ESE + cisplatin group (41.3%) compared with untreated controls (96.6%), whereas cisplatin alone induced a more moderate reduction (70.6%). At first glance, this pattern may appear counterintuitive in the context of enhanced apoptosis. However, Smac/DIABLO is released into the cytosol upon mitochondrial outer membrane permeabilization and may subsequently undergo rapid turnover and degradation. Given the prolonged treatment duration employed in this study (48 h), the reduced detectable Smac/DIABLO levels may reflect efficient mitochondrial release followed by accelerated post-release degradation, rather than diminished pro-apoptotic activity. This interpretation is supported by previous reports demonstrating that sustained apoptotic signaling can be associated with decreased steady-state Smac/DIABLO abundance due to its functional engagement and proteasomal turnover during apoptosis, including via IAP E3 ligase activity (e.g., XIAP- and Livin-dependent ubiquitination of Smac) [23,24].

Importantly, this phenomenon is mechanistically linked to the regulation of IAP proteins, particularly XIAP. In addition to inhibiting caspases, XIAP functions as an E3 ubiquitin ligase that targets mitochondrial-released Smac/DIABLO for proteasomal degradation. Accordingly, XIAP induction may actively contribute to Smac clearance during sustained apoptotic signaling. In line with this mechanism, XIAP and cIAP-1 were markedly reduced under cisplatin single-agent conditions, which may indicate stress-induced suppression of anti-apoptotic defenses. By contrast, XIAP levels were substantially elevated in the combination group (51.1%), exhibiting a nearly threefold increase compared to the untreated control. Although XIAP is classically viewed as anti-apoptotic, its induction in the context of maximal caspase-3 activation and robust p53 signaling may reflect a compensatory but ultimately ineffective survival response. Cancer cells often upregulate XIAP in an attempt to restrain excessive caspase activity; however, when p53-dependent transcriptional programs are fully engaged, XIAP induction may become insufficient to prevent apoptotic progression. Accordingly, XIAP upregulation may represent a compensatory response aimed at degrading released Smac/DIABLO and limiting mitochondrial stress, rather than an effective anti-apoptotic mechanism [23].

BAD, a pro-apoptotic member of the Bcl-2 family, also displayed context-dependent changes in expression. BAD levels were markedly suppressed (by nearly fourfold) under both ESE and cisplatin single-agent conditions, consistent with a survival-oriented cellular response. In contrast, BAD was partially restored in the combination group, reaching 26.6% of maximal expression. As BAD promotes apoptosis by neutralizing the anti-apoptotic proteins Bcl-2 and Bcl-XL and facilitating mitochondrial outer membrane permeabilization, its re-expression under combination treatment suggests reinstatement of mitochondrial susceptibility to apoptosis. This shift is in line with the observed p53 reactivation and robust caspase-3 cleavage, further supporting coordinated engagement of the intrinsic apoptotic pathway in response to combined ESE-cisplatin exposure.

Additional stress regulators revealed a context-dependent modulation of redox control. PON2, an intracellular antioxidant enzyme involved in maintaining redox balance, was preserved under cisplatin exposure, indicating sustained antioxidant support during genotoxic stress. In contrast, PON2 levels were reduced in the presence of ESE, both alone and in combination with cisplatin. The partial preservation of PON2 in the combination group suggests a balanced redox state in which antioxidant demands are met while allowing cisplatin-induced DNA damage signaling to proceed.

HTRA2/Omi, a mitochondrial serine protease that promotes apoptosis by antagonizing IAPs, was nearly depleted in the ESE-treated group, markedly reduced (three- to fourfold) under cisplatin exposure, and partially restored under combination treatment. This expression pattern suggests that ESE alone favors cytoprotective stress adaptation, while suppression of HTRA2/Omi under cisplatin exposure may reflect an apoptosis-resistant state. In contrast, the partial restoration observed with combined treatment indicates re-engagement of mitochondrial apoptotic machinery, consistent with active release and functional utilization of the mitochondrial protein during apoptosis.

HSP27, a molecular chaperone involved in cytoskeletal and mitochondrial stabilization, was modestly reduced under all treatment conditions, with the most pronounced decrease observed in the ESE and combination groups. This pattern indicates a consistent attenuation of HSP27 expression in response to treatment, particularly in the presence of ESE, which may contribute to lowering the apoptotic threshold.

The extrinsic apoptotic pathway displayed a more variable regulation. The adaptor protein FADD was reduced by approximately 50% across all treatment groups, suggesting a general suppression of death receptor signaling. However, Fas/CD95 expression followed a different pattern. Its levels were reduced under both ESE and cisplatin single-agent conditions by approximately 22% and 14%, respectively, suggestive of a possible resistance mechanism to programmed cell death via the extrinsic pathway. Conversely, the combination treatment restored Fas/CD95 expression to nearly untreated control levels, enabling reactivation of extrinsic death receptor-mediated apoptotic signaling.

The observed chemosensitizing and pro-apoptotic effects of Erica spiculifolia extract can be interpreted in the context of its phytochemical composition, which is dominated by flavonol glycosides and phenolic acids with well-documented anticancer and apoptosis-modulating activities. Phytochemical profiling has identified kaempferol 3-O-glucoside (astragalin) and myricitrin as the predominant flavonols in ESE, followed by substantial levels of the flavanone naringenin 7-O-glucoside (5958.96 ± 9.98 ng/mg), alongside chlorogenic, cinnamic, quinic, and gallic acids as the major phenolic acids. The reported biological activities of these major constituents provide a strong mechanistic rationale for the observed chemosensitizing effects of the extract. Rather than acting as a dominant cytotoxic agent, ESE appears to function primarily as a multi-target modulator of apoptosis-related signaling pathways, resulting in re-sensitization of colorectal carcinoma cells to the conventional cytostatic cisplatin.

Astragalin, a major phenolic constituent of E. spiculifolia, has been shown to exert pronounced anticancer activity in colorectal cancer models. A recent in vivo and in vitro study demonstrated that astragalin effectively suppressed colon tumor development in mice through inactivation of the NF-κB pathway, a central regulator of inflammation-driven tumor progression and chemoresistance. In HCT116 colorectal carcinoma cells, astragalin inhibited cell proliferation and migration by downregulating MMP-2 and MMP-9, induced G0/G1 cell cycle arrest via suppression of CDK2, CDK4, Cyclin D1, and Cyclin E, and concomitantly increased the expression of the cyclin-dependent kinase inhibitors p21 and p27. Importantly, astragalin promoted apoptosis by upregulating caspase-3, -6, -7, -8, and -9, p53, and Bax, while suppressing Bcl-2 expression [25]. Myricitrin, another dominant flavonol in ESE, has similarly been implicated in apoptosis induction across cancer models. In HL-60 leukemia cells, myricitrin was shown to disrupt mitochondrial membrane potential, increase DNA fragmentation, and suppress key survival proteins such as RAS, B-RAF, and BCL-2, while activating stress- and apoptosis-associated signaling proteins including p38, pro-caspase-3, pro-caspase-9, and active caspase-3 [26]. These molecular effects are well in line with the apoptosis-related proteomic alterations observed in the present study, particularly the restoration of p53 signaling and caspase-3 activation under combination treatment.

Phenolic acids being present in high abundance in ESE further supports this proapoptotic and chemosensitizing profile. Chlorogenic acid (CGA), one of the dominant phenolic acids identified, has been extensively studied for its anticancer potential in colorectal carcinoma, including in HT-29 cells [27,28]. Chlorogenic acid reduced cell viability of HT-29 cells in a dose-dependent manner and induced cell cycle arrest and apoptosis through upregulation of p21 and p53, suppression of Bcl-2 and NF-κB signaling, and activation of caspase-3/-9 [29]. Notably, CGA has been shown to activate both intrinsic and extrinsic apoptotic pathways, involving modulation of Bcl-2 family members and Fas/CD95 signaling [29], which aligns well with the differential regulation of Fas/CD95 observed in the present proteomic analysis. Quinic acid (QA), another major phenolic acid in ESE, has demonstrated synergistic anticancer activity when combined with cisplatin in squamous cell carcinoma models. In oral cancer cells, QA enhanced cisplatin-induced apoptosis through downregulation of anti-apoptotic genes and attenuation of the Akt signaling pathway, a known contributor to cisplatin resistance in colorectal carcinoma [30]. Gallic acid (GA) has also been reported to exert potent anticancer effects against colorectal carcinoma cells, including through induction of apoptosis in HCT116 cells [31]. Importantly, gallic acid has been shown to synergize with cisplatin in human breast adenocarcinoma cells by enhancing apoptotic signaling while sparing non-malignant epithelial cells, underscoring its potential as a selective chemosensitizer. [32]. Such selective enhancement of cytotoxicity is consistent with the favorable DRI and CI profiles observed for the ESE-cisplatin combination in our study. Furthermore, cinnamic acid (CA), present at high levels in ESE, has also been reported to enhance the cytotoxicity of the chemotherapeutic agent vinorelbine through dual mechanisms, including activation of PTEN-mediated apoptotic signaling and induction of ATG5-associated autophagy [33]. Notably, the presence of an α,β-unsaturated carbonyl moiety in CA is considered critical for its interaction with cellular targets and may underlie its broad bioactivity profile [34].

Furthermore, ESE contains flavonols such as kaempferol-3-O-glucoside (astragalin) and myricetin derivatives that are plausibly positioned to enhance cisplatin responsiveness by converging on stress- and DNA-damage-responsive signaling centered on p53. Prior work in colon cancer models indicates that kaempferol can act synergistically with cisplatin to augment cytotoxicity and apoptotic output, consistent with amplification of damage-response pathways [35]. In parallel, kaempferol has been linked to p53 activation, including phosphorylation events (e.g., Ser15) associated with stabilization and transcriptional engagement of pro-apoptotic programs [36]. Notably, astragalin (kaempferol-3-O-glucoside) has also been reported to modulate p53 and caspase-associated apoptosis-related proteins in cancer cells, supporting a role for glycosylated kaempferol species in reinforcing intrinsic, caspase-dependent apoptotic signaling [37]. Complementarily, myricetin has been shown to inhibit the growth of cisplatin-resistant ovarian cancer cells via a p53-dependent apoptotic pathway, suggesting that myricetin-like constituents may help lower the apoptotic threshold to platinum-induced DNA damage [38,39]. These reports are consistent with a model in which ESE flavonols enhance cisplatin sensitivity by reinforcing p53-dependent checkpoint and apoptotic signaling, enabling efficient downstream caspase activations.

2.5. Results from AO/PI Staining Assay

To complement the proteomic profiling data indicating activation of apoptosis-related pathways, as well as the combination treatment analyses demonstrating synergistic cytotoxicity between ESE and cisplatin (CDDP), apoptosis and cell death were further examined using acridine orange/propidium iodide (AO/PI) fluorescence staining in HT-29 cells (Figure 7). This morphological assay enables direct visualization of nuclear alterations and membrane integrity, allowing discrimination between viable, early apoptotic, and late apoptotic or necrotic cells. By providing qualitative confirmation of chromatin condensation and DNA fragmentation, the AO/PI assay represents a well-validated and widely applied method for morphological assessment of apoptosis, serving as an independent verification of the molecular and functional findings obtained in the preceding experiments.

Figure 7 shows fluorescence micrographs of cells stained with acridine orange (AO, top row) and dual AO/propidium iodide staining (AO/PI, bottom row) under four conditions: Control, ESE, CDDP, and ESE + CDDP. In the naïve control group, HT-29 cells maintained their typical epithelial morphology and adherence capacity, displayed uniform green fluorescence with intact nuclear structure, indicating preserved membrane integrity and high viability. In contrast, cells treated with ESE or cisplatin (CDDP) exhibited a clear increase in yellow to orange fluorescence, characteristic of early apoptotic cells. Within the AO staining regimen, cells demonstrated condensed and fragmented chromatin, visible as yellow/orange nuclear staining (single-stranded DNA, highlighted by pink arrows). Early apoptosis is marked by these nuclear alterations, reflecting DNA condensation and fragmentation while membrane integrity is still partially preserved. CDDP treatment further increases the proportion of orange and red cells, with pronounced nuclear condensation in AO images and strong PI uptake in AO/PI images, consistent with extensive apoptotic and necrotic death. In the combined ESE + CDDP group, green cells are largely absent; most cells display bright orange or red fluorescence with fragmented or shrunken nuclei (pink arrows), indicating maximal induction of apoptosis and irreversible cell death

3. Materials and Methods

3.1. Plant Material and Sample Extraction

Erica spiculifolia aerial parts were collected as previously described [19]. A voucher specimen was deposited at the Herbarium of the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences (SOM) (Voucher specimen No. 179374). E. spiculifolia aerial parts were dried at room temperature. The extraction procedure was carried out with 80% methanol as previously described [19]. We accurately weighed about 1.0 mg of the lyophilized extract, which was dissolved in a mix of 0.1% formic acid/methanol (3/1, v/v) to obtain a solution with an exact 1.0 mg/mL concentration. The solution was filtered on 0.45 µm cellulose acetate filter and 10 µL was injected for analysis. For cell-based assays, the lyophilized extract was reconstituted in dimethyl sulfoxide (DMSO) to prepare stock solutions and further diluted in culture medium to the desired working concentrations.

3.2. Chemicals

The standard substances—rutin, kaempferol, naringenin, fisetin, quercetin, hesperidin, apigenin, luteolin, myricetin, myricitrin, resveratrol, kaempferol-3-O-glycoside, gallic acid, chlorogenic acid, vanillic acid, caffeic acid, ellagic acid, ferulic acid, o-coumaric acid, m-coumaric acid, p-coumaric acid, gentisic acid, protocatechinic acid, o-hydroxybenzoic acid, m-hydroxybenzoic acid, ferulic acid, syringic acid, cinnamic acid, catechin, epicatechin, epigallocatechin, epigallocatechin gallate and epicatechin gallate were purchased from Extrasynthese (Genay, France). Formic acid and acetonitrile of LC-MS grade were obtained from Merck Co., Ltd. (Darmstadt, Germany). All remaining used reagents were of the highest purity available in the laboratory.

3.3. UHPLC-HRMS

The analyses were carried out on Q Exactive^®^ hybrid quadrupole-Orbitrap^®^ mass spectrometer (ThermoScientific Co., Ltd., Waltham, MA, USA) equipped with a HESI^®^ (heated electrospray ionisation) module, TurboFlow^®^ Ultra High-Performance Liquid Chromatography (UHPLC) system (ThermoScientific Co., Ltd., Waltham, MA, USA USA) and HTC PAL^®^ autosampler (CTC Analytics, Zwingen, Switzerland).

3.3.1. Chromatographic Conditions

The chromatographic separations of the analyzed compounds were achieved on Nucleodur C18 Isis (100 × 2.1 mm, 3 µm) analytical column (Macherey-Nagel, Düren, Germany). using gradient elution at 300 µL/min flow rate. The used eluents were: A—0.1% formic acid in water; B—0.1% formic acid in ACN.

3.3.2. Mass Spectrometry Conditions

Full-scan mass spectra over the m/z range 100–1200 were acquired in negative ion mode at resolution settings of 140,000. The mass spectrometer operating parameters used in a negative ionization mode were: spray voltage −4.0 kV; capillary temperature −320 °C; probe heater temperature −300 °C; sheath gas flow rate 25 units; auxiliary gas flow 12 units; sweep gas 2 units (units refer to arbitrary values set by the Q Exactive Tune software Version 2.1) and S-Lens RF level of 50.00. Nitrogen was used for sample nebulization and collision gas in the HCD cell. All derivatives were quantified using 5 ppm mass tolerance filters to their theoretically calculated m/z values. Data acquisition and processing were carried out with XCalibur^®^ ver 2.4 software package (ThermoScientific Co., Ltd., Waltham, MA, USA).

3.3.3. Quantitative Analysis

The concentration of analyzed compounds in samples was determined using external standard calibration mode. The linear calibration curve of each individual substance was constructed in the range from 0.01 ng/mL to 20,000 ng/mL. The quantitative analysis of the analyzed compounds was conducted using 5 ppm mass tolerance filters to their theoretically calculated m/z values and chromatographic retention time

3.3.4. Standards Preparation

Standard stock solutions of each individual compound were prepared in methanol at a concentration of 1.0 mg/mL and stored at −20 ˚C. Working calibration solutions at concentrations in the range from 0.01 ng/mL to 20,000 ng/mL for each individual compound were prepared by serially diluting the standard stock solutions with a mix of 0.1% formic acid /methanol (3/1, v/v). All analyses were performed in triplicate.

3.4. MTT Cytotoxicity Assays

3.4.1. Cell Lines and Culture Conditions

The antineoplastic activity of Erica spiculifolia extract (ESE) was evaluated in human colorectal carcinoma HT-29 cells and compared with the reference chemotherapeutic agent cisplatin, both as monotherapies and in combination. The HT-29 cell line was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ GmbH, Braunschweig, Germany). Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 5% L-glutamine and maintained at 37 °C in a humidified atmosphere containing 5% CO_2_.

3.4.2. Cell Viability Assay

The effects of Erica spiculifolia extract (ESE) and cisplatin on cell viability were evaluated in HT-29 colorectal carcinoma cells using the Mosmann MTT assay, a validated method for assessing cellular metabolic activity and viability. Exponentially growing cells were harvested and seeded into 96-well plates at an appropriate density in a final volume of 100 µL per well. Following overnight incubation to allow cell attachment, the cells were treated with serial five-fold dilutions of ESE and cisplatin in the concentration ranges 180.0–11.2 µg/mL for ESE and 45.0–2.8 µg/mL, respectively. After treatment, the cells were incubated for 72 h under standard culture conditions. At the end of the incubation period, a filter-sterilized MTT solution (5 mg/mL) was added to each well, and the plates were further incubated for 2–4 h to allow the formation of purple, insoluble formazan crystals by metabolically active cells. The resulting formazan crystals were then solubilized using an isopropanol solution containing 5% formic acid. Absorbance was measured at 550 nm using a microplate reader. The recorded absorbance values were corrected by subtracting background absorbance from blank wells containing MTT and solvent only and were subsequently normalized to the mean absorbance of untreated control cells, which was defined as 100% cell viability. The normalized absorbance data were processed using non-linear regression analysis in GraphPad Prism^®^ software (version 8.0) to determine dose-response relationships and IC_50_ values. All MTT viability assays, including combination experiments, were performed in triplicate independent measurements.

3.4.3. Chou–Talalay Method

The establishment and quantitative evaluation of the synergistic interaction between Erica spiculifolia extract (ESE) and cisplatin were performed using the Chou–Talalay method, implemented through CompuSyn^®^ software (ComboSyn Inc., Paramus, NJ, USA). Prior to combination studies, individual dose–response relationships for each agent were determined using the standard MTT assay, as described in the previous section. Synergistic interactions between ESE and cisplatin were subsequently assessed in MTT-based combination experiments employing a variable-ratio treatment design. A fixed concentration of ESE (45 μg/mL) was combined with serial five-fold dilutions of cisplatin, starting from 45 μg/mL, to evaluate the effect of increasing cisplatin exposure in the presence of ESE.

At each experimental point corresponding to the actual treatment concentrations, drug interaction effects were analyzed based on automatically calculated parameters: Combination Index (CI) and Dose Reduction Index (DRI) values. The CI quantitatively defines the nature of drug interactions, where CI < 1 indicates synergism, CI = 1 denotes an additive effect, and CI > 1 reflects antagonism in fixed- or variable-ratio combinations. The DRI–Fa (fraction affected) plots illustrate the fold reduction in the effective dose of each agent when used in combination, with DRI > 1 indicating a synergistic benefit and 0 < DRI < 1 suggesting antagonism. In addition, isobolograms were generated to visually represent interaction profiles and to serve as complementary tools for interpreting the combined therapeutic performance of ESE and cisplatin.

3.5. Proteomic Profiling

A series of immunoassay experiments was performed to characterize treatment-induced alterations in apoptosis-related protein expression following exposure to Erica spiculifolia extract (ESE), cisplatin, and their combination. Changes in the expression of multiple cancer-associated proteins were assessed in response to treatment with equi-inhibitory concentrations corresponding to the respective IC_50_ values (ESE: 40.2 µg/mL; cisplatin: 33.8 µg/mL) and compared with untreated control cells.

Protein expression profiling was carried out using membrane-based sandwich immunoassays according to the manufacturer’s instructions (Human Apoptosis Array Kit, ARY009; R&D Systems, Minneapolis, MN, USA). Signal intensities of individual protein spots were quantified by densitometric analysis using ImageJ software (version 1.8.0) and subsequently converted into absolute protein expression values. Each protein target was assessed in duplicate on the membrane array in accordance with the manufacturer’s assay design, and the corresponding signal intensities were averaged for quantitative analysis. The maximal signal corresponded to the manufacturer-supplied positive control spots on each array membrane and was used as the normalization reference. Proteins showing the most pronounced treatment-induced modulation were further subjected to comparative analysis to elucidate pathway-specific patterns associated with monotherapy and combination treatment.

3.6. Acridine Orange/Propidium Iodide (AO/PI) Staining

Apoptosis and cell death were assessed in HT-29 human colorectal carcinoma cells using acridine orange/propidium iodide (AO/PI) fluorescence staining. Cells were seeded in 6-well culture plates and allowed to adhere overnight under standard conditions. The cells were subsequently treated for 48 h with ESE (45 μg/mL), cisplatin (CDDP, 45 μg/mL), or their combination at the same concentrations.

After treatment, the culture medium was carefully aspirated, and the cells were gently washed with phosphate-buffered saline (PBS) to remove residual medium. A freshly prepared AO stock solution (2.5 mg/mL) was diluted 1:1000 in PBS to achieve a final concentration of 2.5 μg/mL in the wells, and propidium iodide was added immediately before use. The staining solution was applied to the cells and incubated briefly at room temperature in the dark.

Following incubation, the cells were washed twice with PBS to remove excess dye and immediately examined under a fluorescence microscope. Viable cells exhibited uniform green nuclear fluorescence, early apoptotic cells displayed yellow to orange fluorescence corresponding to chromatin condensation and DNA fragmentation, and late apoptotic or necrotic cells showed red fluorescence due to loss of membrane integrity and propidium iodide uptake. Representative images were captured under identical exposure settings for all experimental conditions (60-fold magnification).

4. Conclusions

This study demonstrates that restoring apoptotic competence is a feasible strategy for enhancing platinum-based chemotherapy in colorectal carcinoma. The combination of Erica spiculifolia extract with cisplatin increased antitumor efficacy not by intensifying cytotoxic stress, but by reactivating intrinsic apoptotic signaling. In particular, the combined treatment promoted robust p53 activation in a context where cisplatin alone elicited an incomplete apoptotic response, highlighting p53 reactivation as a central mechanism of chemosensitization. These findings were further supported by acridine orange/propidium iodide fluorescence staining, which morphologically confirmed enhanced apoptosis and cell death following combined ESE-cisplatin treatment. The quantitative synergy analysis revealed a strong superadditive interaction, enabling marked reductions in cisplatin dose across multiple effect levels. Such dose-sparing effects are of particular relevance given the cumulative toxicity associated with platinum-based therapy and support the potential of combination strategies that enhance therapeutic index rather than drug exposure. Notably, the context-dependent activity of the E. spiculifolia extract underscores the importance of evaluating phytochemicals within combination regimens, as their biological effects may differ substantially between mono- and co-treatment conditions. By disrupting apoptotic escape mechanisms triggered by genotoxic stress, the ESE-cisplatin combination provides mechanistic insights into strategies for mitigating chemotherapy resistance. The main compounds kaempferol 3-O-glucoside, myricitrin, chlorogenic acid, and gallic acid hold significance for the enhancement of cisplatin cytotoxicity. Our findings identify Erica spiculifolia as a promising source of natural chemosensitizers and provide a rationale for further investigation into phytochemical-based approaches aimed at restoring apoptosis and improving chemotherapy outcomes in colorectal carcinoma.