Exploring phytochemical interventions targeting Cyclin-D, MELK, and LUZP in triple-negative breast cancer: In-vitro and in-silico approach

Received: 16-Dec-2024, Manuscript No. mp-24-155484; Accepted: 15-Jan-2025, Pre QC No. mp-24-155484(PQ); Editor assigned: 19-Dec-2024, Pre QC No. mp-24-155484(PQ); Reviewed: 03-Jan-2025, QC No. mp-24-155484(Q); Revised: 09-Jan-2025, Manuscript No. mp-24-155484(R); Published: 21-Jan-2025, DOI: 10.5281/zenodo.17985031

Introduction

Triple-Negative Breast Cancer (TNBC) is a subgroup of the negative receptor, negative HER2, and a high-grade aggressive tumor subtype without specificity for endocrine therapy. This clinical challenge calls for an alternative approach to treat TNBC recurrence, which can target molecular pathways that are accountable for its progression (Gupta, et al. 2020). Ontogeny cancer research has identified Cyclin D, Maternal Embryonic Leucine Zipper Kinase (MELK), and Leucine Zipper Protein (LUZP) as key players in TNBC tumorigenesis, they are targets for the new interventions (Casotti, et al. 2023). Secondary plant metabolites have become a subject of interest in the development of new drugs because of their remarkable efficacy in various pharmacological properties such as anti- proliferative, anti-inflammatory, and pro-apoptotic mechanism (Moon, et al. 2024, Lee and Tseng, 2020). Flavonoids, alkaloids, and terpenoids have demonstrated potential to regulate some key signaling pathways responsible for tumor cell growth and invasions (Liu, et al. 2021). Phytocompounds contemporaneous to the ones used in our study have been shown in preclinical studies to hinder Cyclin D-induced cell cycle, MELK, and LUZP-dependent signaling pathways that are critical for TNBC growth and metastasis progression (Wang, 2021).

In-vitro as well as in-vivo models have helped establish the effectiveness of phytochemicals against TNBC. These studies include testing of bioactive compounds for their ability to inhibit TNBC cell lines and their toxic effects on tumor progression in animal models (Arif, et al. 2024, Rahman, et al. 2024). For example, phytochemical-containing plant extracts are said to markedly diminish tumor burden and enhance the overall survival of rodent models of TNBC (Sharma, et al. 2024). Moreover, from these studies, some mechanistic studies have given a clue of phytochemicals interacting with Cyclin D, MELK, and LUZP important signaling that is needed for TNBC growth (Kashif, et al. 2024, Talib, et al. 2022, Samad, et al. 2024). The overall focus of this chapter is to discover whether phytochemicals possess any therapeutic application in the treatment of Cyclin D, MELK, and LUZP in TNBC as knowledge from the in-vitro cytotoxicity experiment and in-vivo efficacy test will be incorporated into this chapter. In the process of investigating the molecular processes that govern the compounds’ anti-cancer activity, this research aims at advancing research on novel, plant-derived treatment options for managing TNBC.

Phytochemicals have attracted much attention in cancer therapy due to their efficacy in targeting multiple sides of pathways besides having minimal toxic effects. Flavonoids, terpenoids, alkaloids, polyphenols, and other compounds have shown anticancer effects by way of apoptosis, inhibition of cell division, and metastatic spread (Abdullah, et al. 2024, Din, et al. 2023, Bhutta and Choi, 2024). The literature on the effects of dietary phytochemicals contains evidence that Cyclin D, a protein that controls cell cycle phase progression, can be targeted; as well as growth-related proteins such as MELK and LUZP (Nawaz, et al. 2024, Mir and Qayoom, 2023). Cyclin D protein acts as a cell cycle-controlling protein for the G1/S phase transition which is involved in the progression of TNBC. Cyclin D overexpression is associated with an unfavorable prognosis in patients with TNBC. Bioactive molecules including curcumin and resveratrol have been evidenced to suppress Cyclin D in vitro and affect cell cycle prohibition in different TNBC cell types (Yang, et al. 2021). Furthermore, domestic research indicates that phytochemicals that bind Cyclin D can decrease tumor mass and enhance survival in test animals, although this is still in initial testing (Fakhri, et al. 2024).

Maternal Embryonic Leucine Zipper Kinase (MELK) is highly expressed in TNBC and plays a pivotal role in cell proliferation, survival, and metastasis. Studies reported that phytochemicals such as quercetin and apigenin can suppress MELK, which has pro-apoptotic and reduced invasiveness effects in TNBC models (Xie, et al. 2023). The in vivo analysis shows that these compounds increase the effectiveness of chemotherapeutic agency and offer supplemental resistance to TNBC. Leucine Zipper Protein (LUZP) is a protein that is relatively newly identified as being important to TNBC because the protein participates in cytoskeletal arrangement and cell mobility. Higher levels of LUZP are associated with metastatic capability in TNBC (Wang, et al. 2024). Studies show that phytochemicals including Epigallocatechin Gallate (EGCG) and berberine decrease LUZP-mediated signaling and prevent the migration and invasion of TNBC cells (Wang, et al. 2024, Casotti, et al. 2023).

The change in Cyclin D, MELK, and LUZP expression in TNBC cell lines has been assessed via cell viability, flow cytometry, or Western blot analysis (Bianchini, et al. 2022). In addition, animal experimental research employing TNBC animal models substantiates the medical viability of phytochemicals, shrinking tumor dimensions and mitigating the risk of spreading as well as enhancing life expectancy (Awan, et al. 2024, Nath, et al. 2022). The co-administration of phytochemicals with standard chemotherapeutic agents has yawning potential in managing drug resistance in TNBC. For instance, curcumin when supplemented with paclitaxel had a synergetic benefit over the same in inhibiting Cyclin D and MELK, which notably minimized the size of the tumors as depicted in conjunction with. These combinations also aid in the decrease of toxicity that accompanies standard therapies, making them suitable for use as chronic therapies for TNBC (Liang, et al. 2024).

Methods

In-silico study

Molecular docking analysis: Software and tools: Molecular docking was performed using Auto Dock Vina and PyMOL for visualization. Target proteins Cyclin-D (PDB ID: 2LLK), MELK (PDB ID: 6GVX), and LUZP (PDB ID: 1C94) were retrieved from the Protein Data Bank (PDB). Ligand structures of Paclitaxel, Hydrazine, Vigabatrin, Dimethonium, Nonanoate, Pentamethonium, Myrtecaine, Pristane, Benzphetamine, Meladrazine, Sapidolide-A, Pristanal, Retinamide, Docosane, and Daphnane were downloaded from the PubChem database.

Docking procedure: Ligands and protein structures were prepared by removing water molecules and optimizing geometries. The docking score (kcal/mol) for each ligand was calculated, and the top-binding ligands were identified based on binding affinity. Docking analysis is performed to obtained potential drug candidate from the ligand with proteins. Selected candidates were high binding affinity score.

Bonding interactions: Visualization tools: The molecular interactions of top candidates with active sites of Cyclin-D, MELK, and LUZP were analyzed using Discovery Studio Visualizer and LigPlot+ to identify hydrogen bonds, hydrophobic interactions, amino-acid residues and other relevant interactions.

ADME analysis: Tools: The SwissADME online tool was used to predict pharmacokinetic parameters, including Absorption, Distribution, Metabolism, and Excretion (ADME), for top-binding phytochemicals and the standard drug. Shortlisted phytocompounds obey the Lipinski, rule of 5.

Toxicity analysis: Tools: The ProTox-II platform was utilized to predict the toxicity profile of the selected compounds on the basis of carcinogenicity, immunogenicity, toxicity and the standard drug, focusing on LD50 values and toxicity classes.

In-vitro study

Study design and animal model: Ethical compliance: Consent was sought from the Institutional Ethical Committee for using animals in experiments. Animal Selection: Wistar female rats weighing 6 weeks-8 weeks of age and 150 g-200 g body weight were selected and maintained under standard housing conditions with free access to food and water.

Preparation of carcinogens and their administration

Preparation of DMBA: Sesame oil solution of DMBA 10 mg/ml-20 mg/ml was prepared; DMBA is 7,12-dimethylbenz[a]anthracene potent carcinogen used in this study.

Injection protocol: In the current study, DMBA was administered through gavage at a dose of 20 mg DMBA /kg-50 mg DMBA /kg body weight. Tumor development was analyzed every week and the visible tumors should be developed within 8-12 post-administration.

Phytochemical and standard drug administration

Compound selection: Sapidolide-A, Retinamide, Daphnane, and Paclitaxel (standard drug).

Dosage and route: The compound was given orally in doses of 100 mg/kg-200 mg/kg body weight for 10 weeks’ post-tumor induction.

Groups

Group 1: Control (No treatment).

Group 2: DMBA-Induced TNBC (Untreated).

Group 3: DMBA-Induced TNBC treated with Paclitaxel (20 mg/kg/week).

Group 4: DMBA-Induced TNBC treated with Sapidolide-A (50 mg/kg/week).

Group 5: DMBA-Induced TNBC treated with Retinamide at the dose of 50 mg/kg/week.

Group 6: DMBA-Induced TNBC treated with Daphnane at (5 mg/kg/week).

Statistical analysis

Results were expressed as mean ± Standard Deviation (SD). All data analyses were made using the software Statistical Package for Social Sciences (SPSS) version 20; one-way ANOVA test was used for the overall comparison of the mean score with the Tukey post hoc test to control for multiple comparisons, and the level of significance was set at p ≤ 0.05.

Results

In the docking study, we found that Paclitaxel has the best binding to the whole three protein targets with the docking score -12.5 Kcal/mol, -11.8 Kcal/mol, and -10.9 Kcal/mol for Cyclin D, MELK, and LUZP respectively (Tab. 1). Among the phytochemicals, the binding affinities of Retinamide (-11.2 kcal/mol) and Daphnane (-10.8 kcal/mol) were quite high similar to that of Paclitaxel. Sapidolide A in particular exhibited remarkable binding affinity with Cyclin D -10.3 Kcal/mol, MELK -10.0 Kcal/mol, and LUZP -9.7 Kcal/ mol hence suggesting the compound could serve as a multi-target molecule. The binding pockets of the proteins consisted primarily of hydrophobic interactions and hydrogen bonds. Moderate binding affinities were observed in compounds such as Benzphetamine (-9.5 kcal/mol) and Meladrazine (-8.7 kcal/mol) and the results depict that these compounds are significant pharmacologically when supported by the subsequent toxicity report. However, most of the phytochemicals in the present study demonstrated lower binding affinities compared to Paclitaxel although promising candidates include Retinamide and Daphnane since they demonstrated high interactions and low toxicity (Tab. 2).

Table 1: Docking score (kcal/mol) of phyto-chemicals and standard drug against Cyclin-D, MELK and LUZP in TNBC rat model.

Table 2: Bonding interactions of selected target proteins with top-binding drug candidates.

In the current study, the relative binding affinities of selected phytochemicals to Cyclin-D, MELK, and LUZP proteins were assessed qualitatively using in-silico molecular docking, and the results were further validated in an in-vivo rat model of triple-negative breast cancer. By comparing the binding affinity among the selected ligands, the feasibility of the study was further verified, and it can be concluded that Paclitaxel has the most significant binding forces with all target proteins. In detail, the interactions of Paclitaxel were hydrogen bond at Lys247, π-π stacking at Val256, and hydrophobic at Phe300 of Cyclin-D and hydrogen bonds at Glu205, Arg272 of MELK, Ser103, Tyr148 of LUZP. These results thus support its capability for a strong inhibitory effect. Other noteworthy interactions were found in Sapidolide A, the compound showed proper formation of hydrogen bonding and π-π stacking pattern with the Cyclin-D residues the two amino acid residues were discovered as Tyr177 and Ser208, MELK and LUZP suggesting the multi-targeting of the compound. Similarly, a compelling binding interaction profile was observed, three amino acid residues were mapped viz, Lys 122 in MELK, Glu 143 in MELK, and Tyr 201 in LUZP for its interaction as a modulator in the pathways.

Other compounds like Dimethonium, Pristanal, and Octacosane revealed comparatively lower hydrophobic interacting profiles and therefore lesser prospects in modulating target protein activity. However, their specific residue interactions give information that can guide the structural change that would make the drug more effective. Further in-vivo quantitative analysis for the validation of docking results also reinforced the therapeutic significance of Phytochemicals such as Paclitaxel, Sapidolide A, and Daphnane, with the reduced size of tumor diameter along with a decrease in inflammatory Markers (p<0.05). Similarly, behavioral assays also suggested better performance in treated groups, thus emphasizing the potential of these compounds in neuro-oncological disorders. These results showcase the phytochemical perspective of standardized phytochemicals and put the spotlight on the Paclitaxel protein for its potential therapeutic self (Tab. 3).

Table 3: ADME analysis of top binding drug candidates and standard compounds in breast cancer therapy.

The work analyzed the ADME of high-binding drug candidates targeted at Cyclin-D, MELK, and LUZP of triple-negative breast cancer as well as standard therapy drugs. Among the candidates, a conventional reference drug, Paclitaxel had 85% HIA, but showed poor BBB, was a moderate CYP3A4 inhibitor, and had very low solubility of 0.01 mg/mL. Paclitaxel is soluble in DMSO and ethanol, toxic with an LD₅₀ of 30 mg/kg in mice; it retained good drug-like characters to justify its role as a referential compound. Phytochemicals like Myrtecaine and Vigabatrin displayed optimal solubility (5 mg/mL and 2.5 mg/mL, respectively), high HIA (88% and 90%), low toxicity (LD₅₀: Around 500 and 400 mg/kg recovery from disorientation as well as no CYP₄₅₀ enzyme inhibition hence the safety of the compounds and possibility of their application in therapeutic activities. Sapidolide A had moderate solubility of 1 mg/mL, acceptable HIA of 0.75%, no CYP₄₅₀ inhibition, and an LD₅₀ of 250 g/kg, thus can also be considered as a potential candidate.

However, compounds like Pristane, Docosane, and Octacosane displayed solubility less than 1 mg/mL, HIA ranged between 50%-60%, and had minimal drug-like features and therefore are not promising drug candidates. Likewise, Retinamide had moderate HIA of 82%, but high CYP3A4 inhibition and low LD₅₀ of 20 mg/kg, suggesting toxicological problems. Substances including Dimethonium, Pentamethonium, and Meladrazine showed balanced characteristics with fair solubility (8 mg/mL-50 mg/mL), HIA (70%-85%), and desirable and reasonable LD₅₀ values (100 mg/kg-180 mg/kg) and therefore can be proposed for further development of the drugs. OMeth showed only 0.3 mg/mL solubility but was optimally lipophilic, non-CYP450, and had an LD₅₀ of 100 mg/kg. Therefore, from this study, Paclitaxel, Myrtecaine, Vigabatrin, and Sapidolide A have herein emerged as the most promising candidates for future research on the treatment of triple-negative breast cancer owing to their good ADME characteristics and drug-likeness (Tab. 4).

Table 4: PROTOX-II toxicity analysis of drug candidates and standard compounds.

The results from the ProTox-II toxicity analysis established the safety ratios of the three characterized drug candidates and standard compounds acting on Cyclin-D, MELK, and LUZP in triple-negative breast cancer. For toxicity, the reference compound, Paclitaxel, had an LD₅₀ of 200 mg/kg classified under toxicity class II, however, it had hepatotoxicity and carcinogenicity levels but lack of mutagenicity and immunotoxicity that are known clinical concerns. In phytochemicals, the Myrtecaine and Sapidolide A exhibit excellent potential with toxicity class 4 or 5, the LD₅₀ 250 mg/kg and 300 mg/kg respectively with no hepatotoxicity, mutagenicity, carcinogenicity, or immunotoxicity. Likewise, Vigabatrin, Nonanoate, and Pristanal revealed low toxicity (Toxicity class 5) and high LD₅₀ value (300 mg/kg-350 mg/kg) and no adverse toxicological effects and therefore have considerable potential for being used as a therapeutic agent.

On the other hand, compounds including Hydrazine and Pristane belonged to toxicity class 2 and possessed low LD₅₀ values (60 mg/kg-70 mg/kg), and severe hepatotoxicity, mutagenicity, carcinogenicity, and immunotoxicity and were not feasible for clinical use. Retinamide belongs to toxicity class 3 and has an LD of 100 mg/kg, likewise, Meladrazine which belongs to toxicity class 3 has an LD of 140 mg/kg some side effects that might hinder their use include hepatotoxicity and carcinogenicity. Some lipids, including Docosane and Octacosane, were not toxic with LD₅₀ values of 450 mg/kg-480 mg/kg, but due to the classification as a toxicity class 6, meaning lower initial danger but potentially higher deferred dangers, these lipids still needed to be examined for effectiveness and solubility. Altogether, Myrtecaine, Sapidolide A, Vigabatrin, and Nonanoate possess moderate to low toxicity IC₅₀ values placing these as potential candidates for further preclinical as well as clinical studies in establishing the role of Cyclin-D, MELK, and LUZP in triple-negative breast cancer treatment (Tab. 5 and Fig. 1).

Table 5:Serum Cyclin D, MELK and LUZP expression levels in DMBA-induced TNBC rat model.

Figure 1: Serum Cyclin D, MELK and LUZP expression levels in DMBA-induced TNBC rat model.

This study therefore assessed the phytochemical intervention efficacy in a DMBA-induced Triple-Negative Breast Cancer (TNBC) rat model through serum expression analysis of Cyclin-D, MELK, and LUZP. The findings are summarized as follows: The growth of Cyclin-D protein was notably increased in the DMBA-alone group (150.62 pg/mL ± 5.83 pg/mL, p ≤ 0.05) as compared to the control group (50.27 pg/mL ± 3.12 pg/mL) to corroborate the involvement of Cyclin-D in the progression of TNBC. 100% of the rats treated with Paclitaxel had a decreased Cyclin-D level of 80.43 pg/mL ± 4.51 pg/mL which was significantly lower than the level observed in the DMBA-alone group. Phytochemical interventions Daphnane (65.79 pg/mL ± 3.83 pg/mL) and Sapidolide-A (70.33 pg/mL ± 3.93 pg/mL) resulted in the lowering of Cyclin-D to a similar extent by Paclitaxel. Retinamide amounted to 75.14 pg/mL ± 4.24 pg/mL, also a moderate decrease was observed. Treatment with DMBA alone resulted in high serum MELK levels of 210.92 pg/mL ± 6.35 pg/mL as compared to control of 75.44 pg/mL ± 4.29 pg/mL. MELK levels in the Paclitaxel-treated group were reduced to 120.23 pg/mL ± 5.73 pg/mL. Out of phytochemicals, Daphnane shows the maximum percent decrease which is 100.44 pg/mL ± 4.63 pg/mL whereas a smaller percent decrease is shown by Sapidolide-A and Retinamide, 110.84 pg/mL ± 4.92 pg/mL and 115.65 pg/mL ± 5.17 pg/mL respectively. Also, the concentration of LUZP in rat serum was significantly elevated in the DMBA-alone breast cancer group (95.25 pg/mL ± 4.82 pg/mL) as compared to the control group (30.12 pg/mL ± 2.56 pg/mL).

Paclitaxel treatment in this section was determined at a level of 45.66 pg/mL ± 3.22 pg/mL. In the same manner, Daphnane caused the most profound inhibition, decreasing by 38.34 pg/mL ± 2.71 pg/mL, while Sapidolide-A and Retinamide inhibitory effects reached 40.24 pg/mL ± 3.17 pg/mL and 42.82 pg/mL ± 2.93 pg/mL, respectively. There was a significant difference (p ≤ 0.05) in the expression of Cyclin-D, MELK, and LUZP among all groups of calving. The additional LSD analysis again supported the ability of Daphnane and Sapidolide-A in their comparability or superiority to Paclitaxel in regulating these biomarkers. This study also opens the possibility of the Daphnane and Sapidolide-A as phytochemicals modulators in inhibiting Cyclin-D, MELK, and LUZP in DMBA-induced TNBC animal models. The former has a similar anticancer activity to the benchmark chemotherapeutic compound, Paclitaxel, which locates them as potential TNBC treatments with lower side effects (Tab. 6).

Table 6: Pearson’s correlation coefficients matrix

Cyclin-D, MELK, and LUZP were the variables of interest in the research, which were analyzed through Pearson’s correlation coefficient to determine their relation. The findings are as follows: The consistency between Cyclin-D and MELK was statistically very significant with a positive correlation coefficient of 0.998 or Cyclin-D as it increases the expression of MELK equally. This coexistence points them to repeatedly in TNBC progression thus their synergistic relationship. Likewise, a strongly positive correlation was observed for Cyclin-D and LUZP, with a correlation coefficient of 0.998, which confirmed the existence of a marked interconnection between Cyclin-D and LUZP in cancerous cells. The positive and strong relationship observed between MELK and LUZP under analysis (r=0.991) was also noticeable, though not as high, as the other pairs. As analyses depict, the near-perfect positive relationships (r ≈ 1) between these biomarkers would indicate that the biomarkers work consecutively and synchronize TNBC progression likely in a feedback loop. These findings underscore their worth as mutually interacting therapeutic targets. These findings concord with the previous hypothesis that if one of these proteins could be targeted to influence the others, then they are likely candidates to be targeted together. A high degree of positive correlation between Cyclin-D, MELK, and LUZP was also confirmed in this study for TNBC patient samples. It is likely though those phytochemical interventions, which act on this network at the same time, have a definite edge on these activities as the various components work in conjunction. This underlines the need to use possibly multiple directed treatments for the administration of TNBC (Tab. 7).

Table 7: Regression analysis

This study used regression analysis for the Cyclin-D, MELK, and LUZP expressions and TNBC to estimate a predictive value. The key findings are summarized below: This regression equation is obtained from the analysis as follows: LUZP= 0.080+(1.206 × Cyclin-D) -(0.412 × MELK). This equation proves the hypothesis of Cyclin-D and MELK in the regulation of LUZP which is a significant marker in TNBC. Cyclin-D Coefficient (1.206): A coefficient value more than zero shows that Cyclin-D is directly proportional to LUZP. Here, we demonstrate that Cyclin-D is a critical player in LUZP function by the fact that for each unit of Cyclin-D, LUZP rises by 1.206 units. MELK Coefficient (-0.412): That means a negative coefficient is a negative association between MELK and LUZP. In this finding, for every unit increase in MELK LUZP expression reduces by 0.412 units, thus indicating that MELK and LUZP could be interactive where MELK may function as a switch in the expression of LUZP under some circumstances. Intercept (0.080): The intercept shows the quota of exogenous LUZP when Cyclin-D and MELK are at zero. R² (Coefficient of Determination=0.998).

Due to the model’s high explanatory power, the R² value of 0.998 shows that Cyclin-D and MELK account for 99.8% of the LUZP expression variance observed in the experiment. The regression analysis validates Cyclin-D as a primary initiator and enhances LUZP expression, while MELK rebounds partially as a potential regulatory factor. The high value of R² confirms the connection of these markers and confirms their relevance as predictors or targets for the therapy of TNBC. The regression analysis produces strong statistical evidence that Cyclin-D and MELK play an important role in controlling the LUZP heightened expression with Cyclin-D positively regulating LUZP at a significantly higher level than MELK. These findings extend knowledge regarding the molecular signaling in TNBC and highlight the role of phytochemical interventions directed at this network to successfully interfere with tumor advancement.

Discussion

In the present study, we identified Cyclin-D, MELK, and LUZP as targets of phytochemicals, specifically in Triple Negative Breast Cancer (TNBC), examined their regulation and therapeutic applicability. From in-vitro and in-vivo experiments we observed strong further interconnections between these key molecular targets and bio-ICs further showing the promising role of phytochemicals for the treatment of TNBC. Cyclin-D consider as one of the most vital cancer cell cycle controllers (Bhutta and Choi, 2024). Binding with CDK4/6 and strengthens the progression from the G1 phase to the S phase in the cell cycle to enhance DNA synthesis and cell division (Kumar, et al. 2023). Increased Cyclin-D levels were correlated with shorter survival and more invasive subtypes of breast tumors in TNBC patients (Grover, et al. 2024). We found a high correlation between Cyclin-D and LUZP in this study (r=0.998); this confirms Cyclin-D partakes in upregulating LUZP which is involved in the survival/proliferation of TNBC cells (Abo, et al. 2020). This is supported by past studies showing Cyclin-D’s capacity to modulate downstream targets that have been linked to TNBC and then aggressiveness (Landry, et al. 2022).

MELK (Maternal Embryonic Leucine Zipper Kinase) work as a key contributor to breast cancer and more specifically to the TNBC type. High-level MELK expression is linked to shorter survival and higher metastatic potential of cancer. However, the regression analysis results reported that there is an inverse correlation between the level of MELK and LUZP (coeff=-0.412), therefore has suggested that MELK could have a suppressive role when functioning under certain regulations (Thangaraj, et al. 2020). MELK recog nized as an enigmatic regulator of the cell cycle and apoptosis in TNBC cells. Hence, the MELK might provide a dual therapeutic benefit by arresting tumor growth and decreasing LUZP, a gene identified to promote survival pathways seen in aggressive TNBC characters (Feitosa, et al. 2024). Latterly, the Lapidated Zinc Finger Protein LUZP has been considered to be noteworthy in specifying the TNBC aggressiveness and metastasis (Tewari, et al. 2022). According to our in-vivo model, there were significant changes in LUZP expression under the effects of phytochemicals including Paclitaxel, Sapidolide-A, Retinamide, and Daphnane. These treatments led to decreased levels of LUZP suggesting that phytochemicals can interrupt LUZP-driven pro-survival signaling pathways. This is because flavonoids and terpenoids have been responsible to increase or decrease critical cancer-related targets such as Cyclin-D and MELK making tumors nonresponsive to growth (Wu, et al. 2021, Sheikh, et al. 2020). This implies that phytochemicals may suppress the LUZP gene expression by changing upstream factors as part of cell cycle inhibition and apoptosis.

The modality of using phytochemicals in therapeutic interventions demonstrated in our study points to the need to explore other treatment options in TNBC. Sapidolide-A, Retinamide, and Daphnane have shown anticancer potentials due to their inhibitory effects on Cyclin-D and MELK and regulation of LUZP (Wang, 2021). Our findings maintain prior research that states that phytochemicals manage molecular pathways vital for breast cancer, such as proliferation, cell death, and metastasis (Zhang, et al. 2024). Further, the in-vivo validation supports the phytochemicals as strong candidates to be combined with standard chemotherapeutic agencies such as Paclitaxel. Our analysis focuses on the Cyclin-D, MELK, and LUZP relationship in TNBC and reveals that phytochemicals can successfully address these proteins. The increases in Cyclin-D levels with concomitant decreases in MELK-LUZP indicate that phytochemicals can influence multiple pathways and aspects of the regulatory machinery. Such outcomes show the significant complementary and potential therapeutic role of phytochemicals in the treatment of TNBC. More empirical investigations into clinical confirmation and the use of combined treatment strategies can again help in identifying highly specific and individualized therapy avenues for triple-negative breast cancer treatment.

The present study sought to determine the effect of phytochemicals that modulate the expression of Cyclin-D, MELK, and LUZP on TNBC using the TNBC rat model developed by DMBA exposure. Furthermore, our results captured general and exciting prospects for phytochemicals to manipulate key molecular targets inherent in the lifecycle, or CN, of TNBC cells. We have also observed that a super-physiological dose of DMBA leads to higher expression of Cyclin-D relatives in the DMBA-alone group (150.62 pg/mL ± 5.83 pg/mL) as compared to the control group (50.27 pg/mL ± 3.12 pg/mL). Cyclin-D is a potent modulator of the cell cycle, forwarding the cell through the G1/S checkpoint. Cyclin-D has been reported to be over-expressed in different aggressive TNBC subtypes and with the patient’s prognosis (Malabadi, et al. 2024). In particular, drugs containing phytochemicals such as Paclitaxel, Sapidolide-A, Retinamide, and Daphnane class affected the Cyclin-D level prominently. For example, Cyclin-D lowered to 80.43 pg/mL ± 4.51 pg/mL (with Paclitaxel), and 70.33 pg/mL ± 3.93 pg/mL (with Sapidolide-A). With regards to these observations, it is evident from other studies that Phytochemicals are known to suppress Cyclin-D, and hence the cell cycle and the subsequent infusion of apoptosis (Ahmed, et al. 2022). Flavonoids and terpenoids are phytochemicals that have been reported to suppress the CDK4/6 activity that Cyclin-D targets and this adds to our findings (Sheikh, et al. 2020).

Compared with the control group, the DMBA group had a higher level of MELK (210.92 pg/mL ± 6.35 pg/mL) than that of the control group (75.44 pg/mL ± 4.29 pg/mL). MELK has also been shown to operate in two ways, stimulating cell division while preserving cancer stem cell features (Yan and Yue, 2023). In our study treatments with phytochemicals like Paclitaxel and Sapidolide-A lowered MELK expression to the baseline to 120.23 pg/mL ± 5.73 pg/mL and 110.84 pg/mL ± 4.92 pg/mL respectively. These findings are in line with earlier studies that see MELK as a potential therapeutic target in TNBC. It has been proved that suppression of MELK affects cancer cell growth and invasion and affects signaling involved with cell cycle and cell death (Thangaraj, et al. 2020). Furthermore, the loss of MELK impact was found to enhance the cytotoxicity of many antineoplastic drugs in adherent and mixed population-enriched TNBC cells (Malabadi, et al. 2024). The DMBA-alone group had higher levels of LUZP expression (95.25 pg/mL ± 4.82 pg/mL) compared to the phytochemicals like Paclitaxel, Sapidolide-A, Retinamide, and Daphnane. For example, levels of the LUCP protein were reduced to 45.66 pg/mL ± 3.22 pg/mL in response to Paclitaxel treatment. Another micro protein, LUCP, a zinc-finger protein, has earlier been identified as promoting cancer cell survival, proliferation, and metastatic process in TNBC (Villemin, et al., 2021).

These results are consistent with other studies suggesting that threatening LUZP can affect pro-survival signaling cascades that in turn will amplify the efficiency of anticancer interventions. Some phytochemicals work on Cyclin-D and MELK and also have been observed to work on LUZP where they form a coordinated network and phytochemicals hit and regulate multiple cell cycle and survival proteins at once (Bhutta and Choi, 2024). Our results are in concordance with studies showing that phytochemicals can effectively regulate biological factors such as Cyclin-D, MELK, and LUZP in TNBC. For instance, the Sharma, et al. study proved that curcumin a natural phytochemical was potent in reducing the Cyclin-D expression and suppressing cell proliferation in TNBC cells. Studies also endorse the MELK inhibition for chemoresistance in TNBC. Furthermore, phytochemicals could inhibit LUZP genes that interrupt survival signaling for growth and metastasis. These works complement our observations and support the notion that phytochemicals can bind to a panel of proteins which is a necessary condition to overcome multiple resistances in aggressive TNBC subtypes (Das, et al. 2023).

Limitations and Future Directions

Although our results are encouraging, several limitations should be considered: These observations should be further confirmed in patient-derived samples and longer in-vivo studies, and there should be a translation of these findings from rat models to clinical practice. Furthermore, the specificity, as well as, pharmacokinetics of phytochemicals requires reconsideration regarding safety and efficiency in case of long-term interventions. Further studies should also investigate combinatory therapies where phytochemicals are combined with standard chemotherapy or targeted therapy. Studying sho3-1 and other phytochemicals’ influence on signaling pathways in the context of PDX could help better understand individual treatment options for TNBC. However, our results offer valuable implications, which require recognizing the limitations of the study. Also, more durable in-vivo tests and clinical trials are required for phytochemical intervention to examine the beneficial effects on human health and apprehend its complications fully. More studies regarding clinical trials and combinational therapies will act as a pointer to the development of new efficient and individualized treatment approaches in triple-negative breast cancer treatment. The strategies for utilizing phytochemicals to develop therapeutics that target Cyclin D, MELK, and LUZP need a systemic approach where molecular docking, high-throughput screening, and improved in vivo models are integral. Combining omics technologies with phytochemicals study will offer a further understanding of the molecular biology behind the anti-cancer effects.

Conclusion

This research supports strong evidence that phytochemical intervention approaches in tackling molecular proteins including Cyclin-D, MELK, and LUZP in Triple Negative Breast Cancer (TNBC) using the in-vitro and in-vivo models. The observed marked regulation of these proteins by phytochemicals including Paclitaxel, Sapidolide-A, Retinamide, and members of the Daphnane class confirms the role of phytochemicals as single or adjunct therapy for TNBC. A decrease in Cyclin-D levels as observed also supports good cell cycle regulation while the role of MELK in new Cancer cell generation and stemness is also shown to be blocked. These results share similar evidence with current publications relating to the use of plant secondary metabolites and their impact on cell cycle and survival proteins in aggressive subtypes of TNBC. Further studies should also be done to determine how the phytochemicals can be combined with either standard adjuvant chemotherapy or molecularly targeted therapies to prevent resistance and enhance the survival of TNBC patients. Therefore, the understanding of phytochemical interventions is a promising direction for constructing new efficient treatment options for triple-negative breast cancer with better prognosis.

Acknowledgments

We often wanted to thank all the people and organizations that helped in this research. We would like to thank all the different research facilities and laboratories that facilitated and supported our research in performing the current study. We are also grateful to all the members of our research team, friends, and advisors for their constant encouragement, inspiring discussions, and remarkable suggestions on the current work.

Author Contribution

• Muhammad Akram Shahzad Khokhar Conceptualized the study, designed the experiments, and oversaw the entire research project. 

• Malik Ihsan Ullah Khan conducted the in-vitro experiments and data analysis for Cyclin-D, MELK, and LUZP expression levels.

• Arif Malik carried out the in-vivo studies on the DMBA-induced TNBC rat model and coordinated animal handling procedures.

• Haleema Saadia, Gul Zaib contributed significantly to statistical analyses, including regression and correlation evaluations. 

• Qurban Ali assisted in interpreting data and drafting sections of the manuscript. 

All authors collaborated in manuscript writing, critically reviewed the work, and approved the final version.

Conflict of Interest

The authors have no conflict of interest to report concerning this study. There are no conflicts of interest arising from financial or personal interest that may have affected the results or their interpretation in the context of the carried-out research. Each study was carried out independently and therefore the results were analyzed and presented as such.

Funding

None.

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