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. 2025 Sep 30;10(40):46542–46553. doi: 10.1021/acsomega.5c02803

Crocetin Impairs the Cytochrome P450 Epoxygenase Pathway toward Potentiating the Therapeutic Efficacy of Paclitaxel in Metastatic Breast Cancer

Diksha Manhas †,‡, Gursimar Kaur †,‡, Mahir Bhardwaj †,‡, Harpreet Kour ‡,§, MD Quasid Akhter ∥, Ashiya Jamwal †,‡, Sucheta Sharma †,‡, Parvinder Pal Singh ‡,§, Swarnendu Bag ‡,∥, Anindya Goswami †,‡,*, Utpal Nandi †,‡,⊥,*
PMCID: PMC12529375  PMID: 41114181

Abstract

Triple-negative breast cancer (TNBC) is the most fatal subtype of breast cancer owing to its aggressive nature and limited treatment strategies. Despite significant advancements in cancer research, targeted therapeutic interventions to mitigate TNBC remain limited. Paclitaxel is heavily relied on as a frontline chemotherapy drug for managing breast cancer, but its treatment is often associated with emerging resistance and the onset of severe adverse effects. In the quest to identify an appropriate combination therapy with a low dose of paclitaxel, we explored crocetin using a highly aggressive mouse carcinoma model of breast cancer. The findings demonstrate that crocetin enhanced the therapeutic efficacy of paclitaxel by reducing the tumor burden and limiting the metastatic lung nodule count. Crocetin, in combination with paclitaxel, markedly altered the expression of key epithelial-mesenchymal transition (EMT) markers and substantially upregulated the pro-apoptotic markers. Concurrent treatment of crocetin and paclitaxel suppressed CYP2J2 expression and decreased epoxyeicosatrienoic acid (EET) levels in the tumor tissue. Dysregulated proteins from untargeted proteomics data indicate that crocetin boosted the antimetastatic efficacy of paclitaxel, possibly via modulating extracellular matrix organization. In combination with paclitaxel, crocetin exerted preventive effects by normalizing the cancer-linked WBC count. Devoid of any observed pharmacokinetic effect of crocetin on paclitaxel, overall results dictate that crocetin has significant potential to boost the efficacy of paclitaxel with a possibly crucial contribution from the CYP2J2/EET axis, leading to restricting tumorigenesis. Thus, elucidating the putative molecular signaling pathway is suggested for this promising combination therapy under the frame of targeted therapeutics to combat TNBC.

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Introduction

Triple-negative (ER-negative, PR-negative, HER2-not overexpressed) breast cancer (TNBC) has unique clinical pathological manifestations. It poses a clinically critical challenge due to its aggressive nature, high recurrence rate, increased likelihood of distant metastasis, and poor overall survival compared to other breast cancer subtypes. Despite significant advancements in anticancer therapeutics, there is a scarcity of targeted therapies for the effective management of this lethal disease. As hormonal therapies (selective estrogen receptor modulator/aromatase inhibitor) and HER2-targeted therapeutics (trastuzumab) are nearly ineffective against TNBC, chemotherapy remains the cornerstone of the treatment. However, a significant hurdle with conventional chemotherapy is the emergence of resistance, leading to often futile cancer management, especially in metastatic cases, with around 90% of therapeutic failures. − Consequently, the use of suitable combination therapy is an attractive and proven approach for more efficacious therapy than individual treatments, leading to management of the ailment efficiently. Thus, combination therapy has substantial potential to reduce the probability of chemoresistance by boosting therapeutic efficacy with/without lowering the standard dose of medication. Among the frequently used standard chemotherapeutic drugs, paclitaxel plays a crucial role in treating solid tumors like breast cancer. Paclitaxel mainly functions as an antimicrotubule agent by facilitating the assembly of microtubules that leads to cell cycle arrest and subsequently inhibits cell replication. However, its clinical applications are restricted due to the development of chemoresistance, which is associated with inadequate response, tumor recurrence, and metastases and represents the primary cause of mortality in breast cancer patients. , Furthermore, paclitaxel treatment is associated with several potential adverse effects, especially myelosuppression , and peripheral neuropathic pain. Researchers are also providing efforts for the effective combination of paclitaxel at its lower dose level for treating breast cancer. ,

In the pursuit of improved efficacy of paclitaxel, we framed the hypothesis that crocetin (a phytoconstituent from the stigma of Crocus sativus Linne and the fruits of Gardenia jasminoides Ellis) could serve as a suitable candidate for combination therapy with paclitaxel. Considering the reported in vitro/in vivo data of crocetin, the tentative reasons are as follows: (a) upregulates the p53/p21 pathway; (b) disrupts topoisomerase II; (c) declines the Bcl-2/Bax ratio; (d) inhibits Cyclin B1; (e) reduces the expression of inflammatory mediators, such as IL-6, and IL-8; (g) downregulates pro-survival genes and multidrug resistance proteins. However, no information is available in the literature to date on the effect of crocetin in combination with paclitaxel.

Therefore, the current investigation mainly aimed to assess the impact of crocetin on the efficacy of paclitaxel in an orthotopic mouse model of breast cancer with the underlying mechanisms.

Results and Discussion

Crocetin Augmented the Paclitaxel Effectiveness to Alleviate Tumor Burden

Paclitaxel is a potent chemotherapeutic drug that is highly effective in treating solid tumors, such as breast cancer. Nevertheless, paclitaxel treatment results in the emergence of resistance to chemotherapy, hence impeding its clinical effectiveness. In this view, we aimed to identify an appropriate combination therapy of paclitaxel to treat metastatic breast cancer. In combination therapy, two or more candidates are used to chemosensitize the cells, making the combination more potent. Moreover, combination in chemotherapy offers an improved response rate over single-agent treatment up to 65% in phase II trials. Therefore, we explored the combination of crocetin and paclitaxel as a neoadjuvant therapy toward any enhancement of paclitaxel’s efficacy, with the potential to overcome drug resistance. The study utilized the 4T1 murine mammary carcinoma model, which is known for its high invasiveness and pronounced tumorigenic potential. This particular model has the potential to spontaneously metastasize from the site of the tumor origin to distant organs. Thus, it is an extensively utilized preclinical study model on spontaneous breast cancer metastasis due to its close resemblance to the tumor growth and metastatic patterns observed in human breast cancer. In the present study, paclitaxel was administered to the animals alone and with crocetin. The images of tumors obtained from animals under different study groups are depicted in Figure A. Variations in the body weight of the animals during the experimental period are illustrated in Figure S1. Tumor size and tumor volume were significantly decreased when treated with only paclitaxel or crocetin compared with the disease control group (Figure B,C). Results are consistent with the previous findings regarding the efficacy of paclitaxel alone treatment in reducing tumor burden at the preclinical level. , In addition to the effects of paclitaxel and crocetin administered individually, we also examined the impact of paclitaxel in combination with crocetin at two different doses (25 and 50 mg/kg). Results demonstrate a significant reduction of the tumor weight compared to the alone paclitaxel treatment at only a crocetin dose of 50 mg/kg. Further, concomitant administration of crocetin with paclitaxel displayed a significant deceleration of the tumor volume compared to the alone paclitaxel treatment. Thus, crocetin displayed the most prominent impact at a dose level of 50 mg/kg to augment the efficacy of paclitaxel by reducing tumor volume. Hence, the outcomes of the current work direct further investigation of the effectiveness of this combination in curtailing lung metastasis.

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Tumor images (A), tumor weight (B), and tumor volume (C) of the various study groups. DC represents the disease control group; P represents paclitaxel alone; C50 represents crocetin (50 mg/kg) alone; P + C25 represents concomitant administration of paclitaxel and crocetin (25 mg/kg); P + C50 represents concomitant administration of paclitaxel and crocetin (50 mg/kg). Data are represented as mean ± SEM (n = 5). Statistical significance level: */**/*** represents p < 0.05/0.01/0.001 in DC vs P or C50; #/##/### represents p < 0.05/0.01/0.001 in P vs P + C25 or P + C50; ns represents statistically insignificant.

Crocetin Intensified the Paclitaxel Efficacy to Counteract Lung Metastasis

Metastasis to the lungs is the characteristic feature of aggressive breast cancer. Hence, a thorough evaluation of the extent of lung metastasis is essential to determine the potency of the antimetastatic drug. In this investigation, the 4T1 murine mammary carcinoma model was used, which is known for its notable invasiveness. While examining the incidence of metastasis in the disease control group, we observed substantial metastatic nodules over the lung tissue surfaces (as depicted by marking red arrows in Figure A). We found a reduction in the lung nodule formation following treatment with paclitaxel or crocetin alone compared to the disease control group (Figure B). The antimetastatic effect of both paclitaxel and crocetin is also reported at the in vitro/in vivo level. − Concomitant administration of crocetin with paclitaxel effectively prevented the occurrence of lung nodule formation in comparison to alone paclitaxel treatment. These findings indicate that crocetin has remarkable potential to inhibit the spread of aggressive tumor cells, hence strengthening the efficacy of paclitaxel in preventing metastasis. Based on the current results, we sought to investigate further the alterations in the key metastatic markers.

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Representative lung images (A) and data on lung nodule formation (B) in the various study groups. DC represents the disease control group; P represents paclitaxel alone; C50 represents crocetin (50 mg/kg) alone; P + C25 represents concomitant administration of paclitaxel and crocetin (25 mg/kg); P + C50 represents concomitant administration of paclitaxel and crocetin (50 mg/kg). Data are represented as mean ± SEM (n = 5). Statistical significance level: */**/*** represents p < 0.05/0.01/0.001 in DC vs P or C50; #/##/### represents p < 0.05/0.01/0.001 in P vs P + C25 or P + C50; ns represents statistically insignificant.

Crocetin Boosted the Paclitaxel Impact to Abrogate EMT and Tumor Metastasis

To gain insight into the enhanced antimetastatic effect of paclitaxel in conjunction with crocetin, Western blotting was performed and analyzed the expression of the epithelial-mesenchymal transition (EMT) markers, namely E-Cadherin, Vimentin, and Snail 1, using the tumor tissue lysates obtained from the various study groups. EMT refers to the process in which epithelial cells, which usually are immobile, undergo molecular reprogramming and phenotypic alterations to become motile mesenchymal cells. E-Cadherin acts as a tumor suppressor by preventing the detachment of epithelial cells from the basement membrane. At the same time, Vimentin, an inducer of EMT, is upregulated during the EMT process and plays a significant role in promoting tumor development and invasion. Snail 1, a transcription factor, suppresses E-Cadherin expression and facilitates the EMT process, making it a key inducer in cancer metastasis. ,

In the current investigation, the combination of paclitaxel and crocetin markedly reduced the protein expression of Snail 1 (0.03 to 0.06-fold) and Vimentin (0.17 to 0.40-fold) in comparison to the alone paclitaxel treatment (Figure ). Following the same combination treatment, E-Cadherin level was substantially elevated (1.82 to 2.55-fold) compared to alone paclitaxel treatment. Overall findings imply that crocetin could markedly promote the antimetastatic efficacy of paclitaxel by modulating EMT markers.

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Western blot analysis (A) and densitometry data (B) of metastatic and apoptotic markers in the tumor tissue of the various study groups. β-Actin expression: endogenous loading control. DC represents the disease control group; P represents paclitaxel alone; C50 represents crocetin (50 mg/kg) alone; P + C25 represents concomitant administration of paclitaxel and crocetin (25 mg/kg); P + C50 represents concomitant administration of paclitaxel and crocetin (50 mg/kg).

Crocetin Ameliorated the Potency of Paclitaxel for Apoptotic Induction

To investigate the effects of a combination of paclitaxel and crocetin on apoptosis, Bax and cleaved Caspase-9 were evaluated using Western blotting in the tumor tissue lysates obtained from the various experimental groups. Bcl-2 family proteins are key regulators of the intrinsic pathway of apoptosis since they facilitate the release of pro-apoptotic mediators from the mitochondria. On the other hand, Bax is recognized as a pro-apoptotic executioner inducing apoptosis by forming mitochondrial permeability transition pores and allowing the release of Cytochrome c. Once Cytochrome c is released, cells are committed to death. However, Caspase-9 is a cysteine-aspartic protease involved in apoptosis. Active caspase-9 functions as an initiating caspase by cleaving and activating downstream executioner caspases, thereby triggering apoptosis. Once activated, Caspase-9 proceeds to cleave and activate effector Caspases-3, -6, and -7, which then cleave various cellular targets to trigger apoptotic cell death. Here, we have shown that concurrent treatment with paclitaxel and crocetin caused a significant increase in the protein levels of Bax (2.30 to 2.75-fold) and cleaved caspase-9 (1.73 to 2.38-fold) compared to those of alone paclitaxel treatment (Figure ). These findings indicate that crocetin can substantially amplify the pro-apoptotic effects of paclitaxel, potentially helping to combat the emergence of chemoresistance.

Crocetin in Combination with Paclitaxel Lowered CYP2J2-Mediated EETs

Cytochrome P450 2J2 (CYP2J2) has been demonstrated to be overexpressed in breast cancer and is responsible for the formation of epoxyeicosatrienoic acids (EETs) from arachidonic acid, a predominant polyunsaturated omega–6 fatty acid. , These EETs are linked with tumor cell proliferation, cell migration, and angiogenesis, thus, play an explicit role in promoting breast cancer and its recurrence. , With this study, we explored the functional relationship between CYP2J2-mediated EET formation and breast cancer. An elevated expression of CYP2J2 and raised levels of EETs are reported in several cancers. − In this investigation, we also observed an enhanced expression of CYP2J2 in the tumor tissue of the disease control group (Figure A). Following concomitant administration of paclitaxel and crocetin, a substantial reduction was observed in the protein expression of CYP2J2 compared to alone paclitaxel treatment. CYPs are the key enzymes responsible for the metabolism of drugs and other xenobiotics in the liver. Despite the limited contribution of CYP2J2 in constituting only 1–2% of the total CYP content in the liver, we additionally checked its protein expression in the liver tissue, and the results displayed a similar line of trend-like tumor tissue (Figure B). In parallel, we also quantitatively measured the level of EETs in the tumor tissue, as the role of CYP2J2-linked EET formation in breast cancer is poorly understood. In the present study, we observed a significant reduction in the level of EETs with the treatment of alone paclitaxel or crocetin. Furthermore, concomitant administration of paclitaxel and crocetin (50 mg/kg) appreciably decreased the EET formation (Figure C). In this regard, research works are ongoing to establish that monitoring of EET levels can be used as a prognostic marker for breast cancer diagnosis. Therefore, the current findings infer that the decline in both CYP2J2 expression and EET levels might help to augment the antitumor efficacy of paclitaxel. Based on the present observation, further investigations are directed to identify the dysregulated proteins behind the potentiation of paclitaxel action.

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Western blot analysis and densitometry data of CYP2J2 in the tumor tissue (A) as well as in the liver tissue (B) of the various study groups. β-Actin expression: endogenous loading control. Alteration in the levels of EETs in the various study groups (C). Data are represented as mean ± SEM (n = 3). DC represents the disease control group; P represents paclitaxel alone; C50 represents crocetin (50 mg/kg) alone; P + C25 represents concomitant administration of paclitaxel and crocetin (25 mg/kg); P + C50 represents concomitant administration of paclitaxel and crocetin (50 mg/kg).

Crocetin Lacked Any Impact on the Pharmacokinetics of Paclitaxel

Concurrent administration of any candidate (phytochemical/drug) alongside prescribed medication can precipitate drug interaction. Hence, before going into further investigation of dysregulated proteins, in parallel, we evaluated the pharmacokinetic interaction between crocetin and paclitaxel using a normal mice model (Figure A,B). Notably, concomitant treatment with crocetin did not significantly affect any pharmacokinetic parameters of paclitaxel. It is well established that CYP2C8 & CYP3A4 are the primary enzymes responsible for paclitaxel metabolism in the liver, , and crocetin is a weak inhibitor of both of these CYP enzymes (IC50 > 30 μM). Therefore, crocetin is unlikely to have any pharmacokinetic interaction with paclitaxel.

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Mean plasma concentration vs time profile and main pharmacokinetic parameters of paclitaxel (A and B) and crocetin (C and D) as alone or in combination. Data are represented as mean ± SEM (n = 5).

In addition to paclitaxel, we also assessed the pharmacokinetics of crocetin in the presence of paclitaxel (Figure C,D). The overall plasma exposure of crocetin was significantly reduced in the presence of paclitaxel. The altered T max and AUC are likely linked to changes in the gastrointestinal transit by paclitaxel, similar to marketed chemotherapeutic drugs. − This study indicates that the enhanced efficacy of paclitaxel is not due to any improved plasma exposure of paclitaxel, suggesting that this combination can be safely coadministered, lacking concern for pharmacokinetic interference.

Crocetin Triggered the Antimetastatic Efficacy of Paclitaxel by Improving ECM Organization

From the above discussion, it is pretty clear that crocetin did not have any pharmacokinetics-linked effect on boosting plasma exposure of paclitaxel, thus improving its efficacy. So, there was undoubtedly crocetin-augmented paclitaxel action via a pharmacodynamic interaction. In this pursuit, the role of EET is straightforward toward tumorigenesis. Consequently, we investigated the impact of CYP2J2 inhibition-mediated lowering of EET formation using an untargeted proteomics approach since there is a gap in knowledge about dysregulated proteins and the involvement of tentative signaling pathways in this axis. The untargeted proteomics data of tumor tissue obtained from our various experimental groups mentioned above were evaluated. The relationship was established based on the comparison between alone paclitaxel or crocetin with the disease control group and combined treatment of paclitaxel and crocetin compared to only paclitaxel or crocetin treatment group. Data of the top 50 dysregulated proteins based on a change of 2.0-fold (log2 fold change of ≥ +1.0 for upregulation & ≤ −1.0 for downregulation) are presented in Tables S1–S4, and corresponding gene ontology analysis data are summarized in Tables S5–S8. Compared to only paclitaxel or crocetin treatment, the untargeted proteomics study data reveals that crocetin and paclitaxel combination adequately augmented vital extracellular matrix (ECM) modulating mediators. Analyzing KEGG and REAC databases, we found constitutive activation of several pathways covering a range of ECM functions (collagen assembly, ECM breakdown, ECM organization, and ECM proteoglycans) that are crucial for maintaining cellular hemostasis and structural integrity. Notably, paclitaxel and crocetin combination rendered a significant upregulation of collagen chains [Col3a1 (P08121), Col1a2 (Q01149)], proteoglycans [Decorin (P28654), Lumican (P51885)], and proteolytic enzymes [Procathepsin L (P06797)]. These dysregulated proteins play a critical role as a fundamental building block in the ECM, ensuring the integrity and strength of various tissues along with their contribution to various cellular processes, including cell adhesion, migration, and proliferation, and making them an important player in cancer biology. The ECM and integrin-related pathways (collagen trimerization, integrin cell surface interactions) are critical in cancer metastasis. In this research, we demonstrate that crocetin’s impact on these pathways possibly helps to stabilize the integrity of the ECM, potentially averting ECM degradation, a technique frequently used by metastatic cells to infiltrate neighboring tissues. For example, the ECM may be strengthened by the overexpression of particular collagen chains and proteoglycans, creating a biochemical and physical barrier preventing tumor cell intravasation. Crocetin might be involved in stalling metastatic processes by modulating integrin function, which impacts tumor cell adhesion, migration, and survival. − Collectively, these results demonstrate that crocetin augmented anti-ECM remodeling properties of paclitaxel in TNBC cells.

Crocetin Combined with Paclitaxel Improved Cancer-Linked Altered WBC Count

WBC count, often elevated during infections, is a nonspecific indicator of inflammation linked to various cancers. WBCs, such as neutrophils, monocytes, and eosinophils, generate reactive oxygen species (ROS) and nitric oxide species (NOS) that are highly reactive molecules. If these ROS and NOS are not adequately neutralized, they can damage cellular proteins, lipids, and DNA. In a number of clinical reports, patients with breast cancer had significantly higher WBC counts. , A similar line of effect was observed in our study, where the disease control group showed a marked increase in WBC count (4-fold), neutrophil count (3-fold), monocyte count (3-fold), and platelet count (2-fold) compared to those of the normal control group (Figure ). Lymphocytes play a role in the host’s immune response, and lymphocytopenia is commonly seen in breast cancer patients. In this current study, we also observed that the level of lymphocytes remarkably declined in the disease control group compared to that in the normal control group. Besides these, reductions in RBC count and hemoglobin (Hb) are commonly observed in breast cancer patients. Similarly, we observed a significant drop in RBC count in the disease control group compared to that in the normal control group. Thus, hematological parameters are useful indicators of breast cancer. Although paclitaxel is an effective anticancer drug, its use is often associated with hematological side effects such as leukocytopenia. In this study, paclitaxel administration resulted in a 46% reduction in WBC count compared to the disease control group. Compared to only paclitaxel treatment, concomitant treatment of crocetin and paclitaxel significantly improved the WBC count (62 to 65%) toward the normal control group. Hence, the current findings suggest that crocetin had a notable normalizing effect on cancer-induced WBC count and, importantly, no exacerbating effect on any hematological parameters in the presence of paclitaxel.

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Effect of Crocetin in Combination with Paclitaxel on main hematological parameters: Total WBC count (A), Total RBC count (B), Hb (C), Platelet count (D), Neutrophils (E), Lymphocytes (F), Monocytes (G), and Eosinophils (H) in the various study groups. NC represents the normal control group; DC represents the disease control group; P represents paclitaxel alone; C50 represents crocetin (50 mg/kg) alone; P + C25 represents concomitant administration of paclitaxel and crocetin (25 mg/kg); P + C50 represents concomitant administration of paclitaxel and crocetin (50 mg/kg). Data are represented as mean ± SEM (n = 5). Statistical significance level: #/##/### represents p < 0.05/0.01/0.001 in NC vs DC; †/††/††† represents p < 0.05/0.01/0.001 in DC vs P or C50; */**/*** represents p < 0.05/0.01/0.001 in P vs P + C25 or P + C50.

Conclusions

Crocetin significantly enhanced the antimetastatic efficacy of paclitaxel when crocetin was given in combination with paclitaxel in an orthotopic mouse model of breast cancer. This was achieved by suppressing the tumor burden and lung nodule formation, downregulating EMT markers, upregulating apoptosis, attenuating CYP2J2 expression, and declining EET levels. Dysregulated proteins from untargeted proteomics data indicate that crocetin boosted the antimetastatic efficacy of paclitaxel via modulating extracellular matrix organization. Additionally, crocetin, in combination with paclitaxel, exerted preventive effects by normalizing the cancer-linked altered WBC count. Devoid of any observed pharmacokinetic effect of crocetin on paclitaxel, overall results dictate that crocetin has significant potential to boost the efficacy of paclitaxel with a possibly crucial contribution from attenuating the CYP2J2/EET axis, leading to restricting tumorigenesis. Thus, elucidating the putative molecular signaling pathway behind pharmacological action involving the CYP epoxygenase pathway is suggested for this promising combination therapy under the frame of targeted therapeutics to combat TNBC.

Experimental Section

Chemicals and Reagents

Phenacetin (purity ≥99%), ammonium formate (MS-grade), and cremophor-EL were obtained from Sigma-Aldrich. (±)­14,15-EET (purity ≥98%), (±)­11,12-EET (purity ≥98%), (±)­8,9-EET (purity ≥98%), (±)­5,6-EET (purity ≥98%), and (±)­14,15-EET-d11 (purity ≥99%) were purchased from Cayman Chemical. Formic acid (MS-grade) was procured from TCI. Acetonitrile (MS-grade), DMSO, ethanol, and methanol (MS-grade) were acquired from Thermo Fisher Scientific. All other chemicals/reagents were of bioreagent grade or above. Ultrapure water was used throughout the analysis (Make: Merck-Millipore; Model: Direct-Q3 System).

Cell Culture and Antibodies

The 4T1 cells were cultured in RPMI-1640 medium enriched with 10% fetal bovine serum, 1% streptomycin, and penicillin-G, and they were grown in culture flasks at 37 °C in a 5% CO2 incubator. E-Cadherin (24E10) rabbit mAb (Cat No. #3195S), Bax (D2E11) rabbit mAb (Cat No. #5023S), Snail 1 (C15D3) rabbit mAb (Cat No. #3879S), Antirabbit IgG HRP-linked Ab (Cat No. #7074S), and Antimouse IgG HRP-linked Ab (Cat No. #7076S) were obtained from Cell Signaling Technology. Cleaved Caspase-9 (Asp353) pAb (Cat No. #PA5–105271) was acquired from Thermo Fisher Scientific. Vimentin mouse mAb (Cat No. #BF8006) was procured from Affinity Biosciences. Rabbit CYP2J2 pAb (Cat No. #MBS822016) was purchased from MyBioSource, while anti-β-Actin mouse mAb (Cat No. #A5316) was obtained from Sigma-Aldrich.

Test Articles

Paclitaxel (purity >98%) was provided by Fresenius Kabi Oncology Ltd. (Kalyani, West Bengal, India) as a gift sample. Crocetin was synthesized from commercially available crocin (TCI, Product no. C1527), which was then purified and characterized using 1H NMR, 13C NMR, & HRMS and the chromatographic purity of crocetin (>96%) with a Trans: Cis ratio of 9:1 was determined by HPLC.

Effect of Crocetin on the Antitumor Efficacy of Paclitaxel

Animal Husbandry and Maintenance

Adult female BALB/c mice, aged 5 to 7 weeks, were housed in a group of 5 animals/cage within the individually ventilated caging (IVC) system. This IVC system was kept in a modern animal room with a controlled housing environment, i.e., a light/dark cycle of 12 h each, room temperature of 25 ± 2 °C, and relative humidity of 50 ± 20%. The animals were provided with unrestricted access to a standard pellet diet and water.

Ethical Prerequisites

The animal experiments were carried out in accordance with the guidelines set by the “Committee for Control and Supervision of Experiments on Animals (CCSEA)” (Government of India, New Delhi, India). The study protocol was approved by our “Institutional Animal Ethics Committee (IAEC)” under approval number 339/83/8/2023.

Dose and Dose Formulation

Paclitaxel was administered at 5 mg/kg, while crocetin was given at two different dose levels, 25 and 50 mg/kg. These doses were chosen based on the previous combination studies reported in the literature. ,,, The paclitaxel dose was given in a solution form, which was prepared using the vehicle of 5% ethanol +5% cremophor-EL +90% normal saline (v/v). The dose of crocetin was prepared as an aqueous suspension containing sodium carboxymethylcellulose (0.1%, w/v). The dose volume was 10 mL/kg. All the dose formulations were prepared freshly just before the experiment.

Study Arm and Treatment Protocol

The efficacy study was conducted according to our previously reported protocol. , For which, first, the mouse mammary 4T1 carcinoma cells (1.5 × 106) suspended in 100 μL of serum-free RPMI media and matrigel (1:1) were inoculated subcutaneously into the second right mammary fat pad of each mouse to develop a tumor. Once the palpable tumor developed after around 1 week of tumor initiation, animals were randomly separated into five groups, each with five animals. The groups were categorized as follows: Group-1, which received the vehicle of paclitaxel; Group-2, which received the treatment of only paclitaxel (5 mg/kg); Group-3, which received the treatment of only crocetin (50 mg/kg); Group-4, which received the concomitant treatment of paclitaxel (5 mg/kg) + crocetin (25 mg/kg); Group-5, which received the concomitant treatment of paclitaxel (5 mg/kg) + crocetin (50 mg/kg). Group-1 served as the disease control group (tumor control). In parallel, an additional group of normal female BALB/c mice was maintained without any treatment, which served as a normal control group and was used to monitor the alterations in the disease control group. The body weight of these animals was recorded daily before administering the dose. Paclitaxel dose was administered through the intraperitoneal route on alternate days for an experimental period of 10 days, while crocetin was given daily via the oral route. After the treatment period was completed, the animals had a 12 h overnight fasting period. After which, i.e., on the 11th day, the blood samples were obtained from the retro-orbital plexus of each animal. Subsequently, the animals were euthanized using carbon dioxide, followed by cervical dislocation to sacrifice. The organs/tissues, such as the liver and tumor, were then collected, cleaned with normal saline, dried with tissue paper, and weighed. The tissues were rapidly frozen using liquid nitrogen and stored at −80 °C in a deep freezer.

Evaluation of Tumor Weight and Tumor Volume

To determine the weight of the tumor tissue, an analytical balance (Make: Mettler Toledo; Model: XS205) was used, while tumor volume was calculated using the following formula: 0.5 × Length × Width2.

Assessment of Metastatic Lung Nodules

To evaluate the metastatic potential of 4T1 mammary carcinoma cells, the lung nodules were observed and quantified using an inverted microscope (Make: Nikon; Model: ECLIPSE TS100).

Estimation of Protein Expression in the Tumor and Liver Tissue

The protein expression of metastatic and apoptotic markers (Vimentin, E-Cadherin, Snail 1, Bax, cleaved Caspase-9) in the tumor tissue was assessed using the standard immunoblotting protocol of Western blotting. , For this, a small portion of tumor tissue was weighed in equal proportion from each animal, and then the pooled tissue homogenate was prepared for Western blot analysis, where protein bands were monitored in the Chemidoc system (Make: Syngene; Model: G-Box) and densitometry analysis was done by ImageJ software. Besides these metastatic and apoptotic markers, protein expression of CYP2J2 was assessed in the tumor and liver tissues.

Quantification of EET in the Tumor Tissue

To determine the concentration of EET in the tumor tissue, first, a small portion of tumor tissue was weighed in equal proportion from each animal, and then, the pooled tissue homogenate was prepared for each study group. The tissue homogenate (350 mg/mL) was prepared by using a solvent mixture of methanol and water (80:20) containing 0.2% formic acid (v/v). Following this, it was processed with acetonitrile containing internal standard, followed by vortex-mixing for 2 min and then centrifuged at 18,626g for 10 min at 4 °C. Afterward, the supernatant was decanted into the inner vial and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantitate the levels of EETs, where pooled samples were run in triplicate for each study group.

SWATH-Based Untargeted Proteomics of tumor tissue

The untargeted proteomics study was carried out following earlier reported and in-house standardized protocols. − Briefly, the steps are as follows: (a) preparation of pooled tumor tissue homogenate in RIPA buffer solution using a small portion of tumor tissue in equal proportion from each animal, (b) Extraction of proteins by precipitation with chilled acetone, (c) Prepare resuspension of protein pellets using Tris-HCl with urea at pH 8.5, (d) Estimation of protein content by Bradford assay, (e) Perform the reduction (25 mM of dithiothreitol at 56 °C), alkylation (55 mM of iodoacetamide at room temperature), and trypsin digestion (16 h at 37 °C) of protein, (f) Desaltation of peptides by Ziptip C18 cartridge, (g) Drying of peptides in vacuum concentrator, and (h) Bioanalysis of tryptic peptides by a quadrupole-TOF hybrid mass spectrometer (Make: Sciex, Model: TripleTOF 6600) coupled to an Eksigent-LC system, (i) Analysis of data by Spectronaut software using the processing settings for peak extraction: maximum of 10 peptides per protein, 5 transitions per peptide, >95% peptide confidence threshold and 1% peptide FDR, (j) Normalization of data and report the differentially expressed proteins using a fold change of 2.0-fold or log2 fold change of ≥ +1.0 for upregulation and ≤ −1.0 for downregulation. Afterward, gene ontology analysis was done. We elucidated the functional profiling of the significantly altered 50 proteins each (upregulated/downregulated) and the significance value of pathway search from different databases (KEGG & REAC) was considered −log10 p-value >1.3. Statistical analysis will be performed by using Microsoft-Excel & R platform.

Impact of Crocetin on Paclitaxel-Induced Hematological Alterations

To assess the hematological impact of paclitaxel, we examined the alterations in the key hematological markers in the aforementioned study groups. For this, blood samples were collected into microcentrifuge tubes containing an aqueous 10% EDTA solution (w/v) as an anticoagulant and analyzed using an automated hematology analyzer (Make: Horiba; Model: Yumizen H500).

Influence of Crocetin on the Pharmacokinetics of Paclitaxel

Animal Model

Adult female BALB/c mice, aged 5 to 7 weeks, were used to perform this study. Animal husbandry and its maintenance are already described above. The study protocol to carry out the pharmacokinetic studies was approved by the IAEC under the approval number 339/83/8/2023.

Study Design

The pharmacokinetics of paclitaxel were studied with and without crocetin using a normal mouse model. For this, seventy-five animals were randomly separated into three groups comprising 25 animals per group. Each group was subsequently divided into five subgroups, each consisting of five animals (n = 5), to collect blood at ten different time points using a sparse sampling technique. Group-1 was treated with paclitaxel (5 mg/kg); Group-2 was treated with crocetin (50 mg/kg); and Group-3 was treated with a combination of paclitaxel (5 mg/kg) and crocetin (50 mg/kg). The dose formulations and the routes for dose administration were the same as those mentioned above. Blood samples were collected from the retro-orbital plexus of the animals at various time points, i.e., 0 h (predose), 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h, into the microcentrifuge tubes containing an aqueous solution of 10% EDTA (w/v). Afterward, each blood sample was centrifuged at 18,626g for 10 min to obtain 50 μL of plasma and was stored at −80 °C in a deep freezer.

Bioanalysis

Each plasma sample (50 μL) was processed using acetonitrile (200 μL) containing an internal standard. Afterward, the sample was vortex-mixed for 2 min and then centrifuged at 18,626g for 10 min at 4 °C. The supernatant was then decanted into the inner vial and analyzed using LC-MS/MS. , The matrix-match calibration curves were prepared by spiking a known amount of paclitaxel/crocetin into the blank plasma to quantify in the particular matrix.

Data Analysis

Plasma concentration data for paclitaxel and crocetin with respect to time were fitted into the noncompartmental model to calculate different pharmacokinetic parameters.

Statistical Analysis

An unpaired t-test calculated the statistical significance through an online t-test calculator (GraphPad Prism). The p-values of less than 0.05, 0.01, and 0.001 were considered statistically significant.

Supplementary Material

ao5c02803_si_001.pdf (440.2KB, pdf)

Acknowledgments

D.M., M.B. & G.K. and A.J. & S.S. are thankful to CSIR, UGC-NFSC and DST Inspire (New Delhi, India) for providing their research fellowship. A.G. is grateful to Dr. Avinash Bajaj (Regional Center for Biotechnology, Faridabad, India) for providing cell lines. S.B. gratefully acknowledges the LC-MS/MS facility's support from CSIR-IGIB (New Delhi, India) for proteomics study.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c02803.

  • Body weight of animals in the efficacy study; untargeted proteomics-based upregulated and downregulated proteins/genes data and gene ontology analysis data (PDF)

D.M. contributed to investigation, methodology, formal analysis, and writingoriginal draft; G.K. contributed to investigation; M.B. contributed to investigation; H.K. contributed to investigation; M.Q.A. contributed to investigation; A.J. contributed to investigation; S.S. contributed to investigation; P.P.S. contributed to supervision; S.B. contributed to supervision and formal analysis; A.G. contributed to supervision; U.N. contributed to conceptualization, supervision, and writingreview and editing.

This research was supported by the Council of Scientific and Industrial Research (New Delhi, India) under the internal budget head of MLP21006. UN also acknowledges the Department of Science and Technology (New Delhi, India) for the required support from Bose Institute toward this publication.

The authors declare no competing financial interest.

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