Antitumor potential of hydroethanolic extract of Neolamarckia cadamba on p53 expression and CA 15-3 levels in DMBA-induced mammary carcinoma in rats

Introduction

Breast cancer is a major global health concern with diverse molecular subtypes, complex risk factors, and varied clinical outcomes, requiring improved understanding for effective prevention and treatment. Continued research is essential to refine targeted treatments and improve long-term patient management [1] . The incidence of breast cancer can rise by up to 40%, or 3 million cases annually, worldwide, and the number of fatalities from the disease might rise from 685,000 in 2020 to 1 million in 2040, accordingly to the journal ''The Breast'' [2] . In outbred Sprague Dawley rats, the polycyclic aromatic hydrocarbon-7,12dimethylbenzanthracene (DMBA) is frequently utilized to cause breast tumors [3] . DMBA is a carcinogenic compound whose tumor-inducing effects are influenced by ovarian hormones. The DMBA-induced rat model is widely used because it produces neoplastic changes such as intraductal proliferation and mammary intraepithelial neoplasia, which closely resemble human ductal carcinoma in situ [4] . The p53 tumor suppressor protein, sometimes referred to as the ''Guardian of the Genome'' is encoded by the TP53 gene. Its main function is to protect the integrity of the cell's DNA. Many malignancies have somatic TP53 mutations, which can result in loss of function, dominant negative, or gain-of-function [5] . By controlling the cell cycle, especially at the G1 to S checkpoint, TP53 helps preserve genomic stability by preventing the replication of damaged DNA. p53 is activated in response to cellular stress or DNA damage, and it can momentarily stop cell division to facilitate repair. It encourages programmed cell death to stop aberrant cell survival if the harm is irreparable. Under normal conditions, p53 levels remain low due to continuous degradation, but they rise rapidly in response to cellular stress [6] . CA 15-3 (Cancer Antigen), also known as MUC1, is the most widely used blood marker for breast cancer. In cancer, the large transmembrane glycoprotein MUC1 is frequently overexpressed and glycosylated improperly. At present, Carbohydrate antigen 15-3 (CA 15-3) is primarily utilized for early detection of recurrent breast cancer and for monitoring therapy in patients with advanced stage of disease.

In a prospective analysis with 368 participants, the study showed that individuals with high preoperative levels of CA 15-3 (>30.4 U/mL) performed worse than those with low levels of the marker [7] . Many people with long-term illnesses, including breast cancer, often turn to herbal remedies as supportive therapy. In fact, over half of cancer patients currently use some form of complementary or alternative medicine along with conventional treatment. A member of the Rubiaceae family, Neolamarckia cadamba is generally known as "Kadamba" in Ayurveda. In the ancient Indian medical system known as Ayurveda, this evergreen tropical tree has substantial therapeutic value. The stem bark, leaves, root bark, flowers, seeds, heartwood, and other sections of Neolamarckia cadamba are a veritable gold mine of phytoconstituents. These bioactive substances include fatty acids, alkaloids, terpenoids, flavonoids, and coumarins. Their documented pharmacological actions are diverse and include anti-inflammatory, anticancer, antidiabetic, antioxidant, and wound-healing properties. There are a lot of different botanical roles that Neolamarckia cadamba can play [8] . The search for plant derived anticancer therapies has intensified in recent years, highlighting the potential value of traditional medicinal plants' unexplored therapeutic properties. Neolamarckia cadmba is one such plant that has gained attention due to its reported pharmacological activities. Based on this, the present study evaluates the hydroethanolic extract of stem bark of Neolamarckia cadamba (HENC) for its influence on key biomarkers associated with breast cancer in DMBA induced mammary tumor model in Sprague Dawley (SD) rats. Particular emphasis is placed on CA 15-3, a commonly elevated serum marker in breast cancer, and p53 gene involved in cell cycle control, DNA repair, and apoptosis.

Materials and Methods

Chemicals and reagents

The chemicals and reagents used in the study included 7,12dimethylbenz(a)anthracene (DMBA) procured from Sigma Pvt. Ltd.; Tamoxifen citrate (Caditam-20) obtained from Cadila Healthcare; Petroleum ether (60-80 °C, AR grade) supplied by Loba Chemie Pvt. Ltd.; Absolute ethanol (99.9% AR grade) from S D Fine Chem Limited; and a Rat CA15-3 ELISA kit (Catalogue No. ER0789, 98T) obtained from Fine Biotech Co., Ltd.. All other chemicals and consumables used were of analytical grade and procured from standard commercial suppliers.

Collection of plant material and extract preparation

Stem bark of Neolamarckia cadamba was collected from the university library of Maharashtra Animal and Fishery Sciences University, Seminary Hills, Nagpur. The plant material was authenticated by a professional botanist from Rashtrasant Tukdoji Maharaj Nagpur University. The collected bark was shade-dried, powdered, and defatted using petroleum ether. The dried material was then extracted with 70% ethanol using a Soxhlet apparatus. The obtained extract was concentrated, dried, and stored in airtight conditions until further experimental use.

Experimental animals

Induction of mammary tumor

Mammary tumors were induced by a single subcutaneous administration of 7,12-dimethylbenz[a]anthracene (DMBA) at 20 mg/kg body weight in the left flank region. Tumor development was confirmed after eight weeks using ultrasonography, and tumor-bearing rats were included for further study.

Experimental design and treatment

Animals were randomly divided into five groups (n = 10 per group): T1 (normal control), T2 (positive control), T3 (standard treatment with tamoxifen at 3.3 mg/kg body weight, orally), and two treatment groups, T4 and T5, which received the hydroethanolic extract of Neolamarckia cadamba at doses of 500 and 600 mg/kg body weight, respectively, administered orally for 30 days (Table 1). were added to respective wells, followed by incubation at 37 °C for 90 min to allow antigen binding. After incubation, the contents were discarded and wells were washed twice with wash buffer. Subsequently, 100 µL of diluted biotin-labeled anti-CA15-3 detection antibody was added and incubated at 37 °C for 60 min, followed by three washes with brief soaking. Then, 100 µL of diluted HRP-streptavidin conjugate was added to each well and incubated at 37 °C for 30 min, followed by five washing cycles to remove unbound conjugate. Colour development was achieved by adding 90 µL TMB substrate solution and incubating the plate in the dark at 37 °C for 10-20 min until adequate colour formation occurred. The reaction was stopped by adding 50 µL stop solution, producing a blue-to-yellow colour change, and absorbance was measured immediately at 450 nm using a microplate reader. Mean absorbance values were calculated after subtracting blank readings, a standard calibration curve was plotted, and CA15-3 concentrations in samples were determined from the curve and expressed as pg/ml.

Immunohistochemical assessment of p53

Immunohistochemistry (IHC) of p53 was performed on paraffin-embedded mammary tissue sections which were initially placed on a hot plate at 55-60 °C for 10-15min to remove excess paraffin, followed by three washes in xylene for 5 min each. The sections were then rehydrated through three changes of absolute alcohol (100%) for 5-8 min each, followed by 90% and 80% alcohol for 6-8 min each, and washed three times with phosphate buffered saline (PBS).

Endogenous peroxidase activity was blocked using 3% hydrogen peroxide, after which sections were washed 3-4 times with distilled water and then with PBS. Antigen retrieval was carried out using Tris-EDTA buffer (10 mM Tris base, 1mM EDTA, 0.05% Tween-20, pH 9.0) by microwave heating in four cycles of 5 min each with a 1min interval between cycles; any loss of buffer volume during heating was compensated with distilled water. The slides were allowed to cool, rinsed with distilled water and washed with PBS. Non-specific binding was blocked by incubating the sections with 0.5% bovine serum albumin (BSA) for 30 min at room temperature. After removal of excess BSA, sections were incubated with primary antibody against p53 (Anti-p53 antibody, Abcam; clone PAb240, catalog no. ab26) at a dilution of 1:400 for 1 h at room temperature or overnight at 4 °C. The sections were then washed with PBS twice for 4 min each and incubated with the appropriate secondary antibody (EnVision FLEX +, Mouse, High pH; Dako catalog no. K800221-2) for 60 min. Following two PBS washes (4 min each), immunoreactivity was visualized using a ready-to-use DAB (3,3′-Diaminobenzidine) substrate-chromogen solution for 5 min. The slides were rinsed under running tap water, counterstained with hematoxylin for 3 min, washed and differentiated using 1% acid alcohol. Finally, the sections were dehydrated through graded alcohols (80%, 90%, and 100%), cleared in three changes of xylene for 5min each, and mounted with DPX (Distrene-Plasticizer-Xylene). Scoring was performed according to the criteria presented in Table 2.

Statistical analysis

Experimental data were analyzed using a randomized block design, and statistical significance was interpreted accordingly.

Results

Immunohistochemistry of p53 gene in mammary tissue

The expression of p53 gene in mammary gland tissue was assessed using immunohistochemical analysis (IHC). Nuclear, nucleo-cytoplasmic staining patterns, staining intensity, and the proportion of positively stained cells were used to evaluate the p53 immunoreactivity (Table 3)

The positive immunoreactivity appeared as a dark brown coloration, indicating positive DAB (3,3′-Diaminobenzidine) staining, whereas negative cells showed blue nuclear staining due to hematoxylin counterstain. T1 group (Normal control) showed no expression of p53 and scored as 0, in T2 group (Positive control) intense nuclear expression was evident in neoplastic epithelial cells of solid tumor regions. Furthermore, the stromal cells scattered throughout the tumor masses showing nucleo-cytoplasmic expression. The staining intensity and extent corresponded to score 3, indicating severe p53 expression, which is characteristic of breast tumor and reflects the accumulation of mutant p53 protein due to uncontrolled cell proliferation and genomic instability. Similarly, T3 (Standard control) showed mild nuclear expression of p53 in the neoplastic cells compared to T2 group confirming the effectiveness of tamoxifen in suppressing tumor progression which was graded as score 1, indicating downregulation. In T4 group (HENC @ 500 mg/kg BW), mild nuclear and cytoplasmic expression of p53 protein was observed in neoplastic cells of mesenchymal origin indicating downregulation of p53 (graded as score 1) comparable to tamoxifen group (T3) suggesting a decline in mutant p53 accumulation, likely due to decreased tumor burden and restoration of normal cellular regulation. In the T5 group (HENC @ 600 mg/kg), moderate expression of p53 was observed in neoplastic cells which is graded as score 2. Figure 1 depicts immunohistochemical expression of p53 in all experimental groups. Normal control (T1) 0 2.

Positive control (T2) 3 3.

Standard control Tamoxifen 3.3 mg/kg (T3) 1 4.

Neolamarckia cadamba @ 500 mg/kg (T4) 1 5.

Neolamarckia cadamba @ 600 mg/kg (T5) 2 3. The T1 group (normal control) showed CA15-3 level of 36 ± 2.49 pg/ml, indicating a normal physiological concentration. On the other hand, CA15-3 levels were significantly elevated in the T2 group (positive control) 67.03 ± 1.58 pg/ml, indicating an increased tumor burden and disease progression. The T3 group (standard control) showed a significant decrease in CA15-3 levels of 43.3 ± 2.3 pg/ml, following treatment with tamoxifen @ 3.3 mg/kg demonstrating its therapeutic efficacy. Among the treatment groups, T4 group (HENC @ 500 mg/kg) showed near normal concentration of CA 15-3 of 37.12 ± 2.87 pg/ml indicating most pronounced antitumor effect even comparable to or better than standard group. CA15-3 levels in T5 group (HENC @ 600 mg/kg) were 45.71 ± 2.32 pg/ml similar to standard control group representing moderate efficacy.

Results and Discussion

The present study investigated the effect of Neolamarckia cadamba extract on p53 gene expression and CA 15-3 protein levels in DMBA induced mammary tumor in SD rats. The results demonstrated a marked alteration in p53 expression in mammary tissues following treatment with Hydroethanolic extract of Neolamarckia cadamba (HENC), indicating its potential antitumor activity. Among treatment groups, T4 group (HENC @ 500 mg/kg BW) showed better results analogous to tamoxifen group (T3), with both groups exhibiting lower p53 expression compared to T5 group (HENC @ 600 mg/kg BW). This suggests that by reducing DNA damage and promoting cell cycle regulation, Neolamarckia cadamba may prevent the growth of tumors. IHC is useful and reliable technique for determining p53 expression in routine pathology. Abnormal p53 IHC patterns show a strong correlation with Tp53 mutations and are considered crucial prognostic and surrogate markers for tumour characterization [9] . These results are consistent with Amin et al., (2009) who found a significant downregulation of p53 expression in treated groups and strong positive p53 immunostaining in the DMBA control group when compared to the normal control group. This altered expression pattern of p53 in DMBA induced mammary carcinogenesis reflects the accumulation of mutant p53 protein, which is commonly detected by IHC [10] . p53 is mutated in nearly half of the human cancers, and in approximately 80% of triple negative breast cancer (TNBC) cases, contributing to tumor aggressiveness and metastatic potential. In TNBC, positive p53 IHC expression has been associated with adverse clinicopathological features such as higher histological grade, presence of vessel tumor emboli, reduced disease free and overall survival and p53 expression may function as an independent prognostic factor indicating a worse clinical outcome [11] . Approximately 60% positive nuclear expression of p53 was observed in the mammary tissues of DMBA control rats, whereas negative p53 staining in normal mammary tissues. These results provide more evidence that p53 overexpression is a characteristic of mammary tumours [12] . The agreement between the current findings and earlier research emphasizes the sensitivity and dependability of immunohistochemistry as a method for detecting alteration in p53 expression during tumor growth and interventions. The presence of diverse bioactive phytoconstituents such as alkaloids, flavonoids, glycosides, steroids and tannins in Neolamarckia cadamba was confirmed in a study and insilico studies also revealed that some phytoconstituents exhibited strong affinity towards HER2(erb2) receptor, suggesting that Neolamarckia cadamba possesses anticancer potential against breast cancer [13] . Quercetin, a flavonoid may reduce the accumulation of mutant p53 protein in breast cancer cells by interfering with its translation rather than its gene transcription. This decline in p53 protein levels is associated with decreased cellular proliferation and alterations in cell cycle progression [14] . However, presence of quercetin in the ethanol extract of Neolamarckia cadamba was confirmed based on HPTLC analysis [15] . Preoperative serum CA15-3 levels have a substantial predictive value and elevated concentrations are correlated with higher tumor grade, larger tumor size, and advanced pathological stage [16] . Cancer Antigen 15-3 (CA 15-3) is an FDA-approved breast cancer biomarker used for monitoring treatment response with elevated serum levels correlating with advanced disease stage, larger tumor size (17). The results of the present study indicate that CA15-3 level significantly increased in mammary carcinoma group and decreased following treatment, with T4 group showing the best therapeutic response among treated groups (p < 0.001). It has been documented by Dharmendra et al., 2014 that CA 15-3 levels were decreased in tumor bearing rats after treatment with herbal medications, an effect attributed to the presence of flavonoid components known for their antineoplastic qualities. Anticancer potential of Neolamarckia cadamba as evidenced by its invitro cytotoxic activity against MCF-7, A549 and HepG2 cell lines [19] . Overall, this study suggests that the hydroethanolic extract of Neolamarckia cadamba possesses promising antitumor potential by modulating p53 expression and reducing CA 15-3 levels in DMBA-induced mammary carcinoma. However, further detailed molecular and clinical investigations are required to confirm its therapeutic efficacy and elucidate the underlying mechanisms.

Conclusion

The present study demonstrated that HENC exhibited notable antitumor activity. It resulted in reduced dp53 immunoexpression and decreased serum CA 15-3 levels, indicating suppression of tumor progression. Among the treatment groups T4 group (HENC @ 500 mg/kg body weight) showed effects comparable to the standard drug tamoxifen. These findings suggest that presence of bioactive phytoconstituents could be responsible for its anticancer potential. Overall, the study supports the therapeutic promise of this plant as complementary approach in breast cancer management. Further detailed molecular, toxicological, and clinical studies are required to validate its efficacy and safety.

References

1. Xiong X, Zheng LW, Ding Y, Chen YF, Cai YW, Wang LP, et al. Breast cancer: Pathogenesis and treatments. Signal Transduction and Targeted Therapy. 2025;10(1):49.
2. Arnold M, Morgan E, Rumgay H, Mafra A, Singh D, Laversanne M, et al. Current and future burden of breast cancer: Global statistics for 2020 and 2040. The Breast. 2022;66:15-23.
3. Russo J, Russo IH. Experimentally induced mammary tumors in rats. Breast Cancer Research and Treatment. 1996;39:7-20.
4. Fidianingsih I, Aryandono T, Widyarini S, Herwiyanti S. Profile of histopathological type and molecular subtypes of mammary cancer of DMBA-induced rat and its relevancy to human breast cancer. Open Access Macedonian Journal of Medical Sciences. 2022;10(A):71-78.
5. Hernández BLJ, El-Deiry WS. Tumor suppressor p53: Biology, signaling pathways, and therapeutic targeting. Biochimica et Biophysica Acta-Reviews on Cancer. 2021;1876(1):188556.
6. Dokunmu TM, Opara SC, Awani OU. p53 gene expression and nitric oxide levels after artemisinincaffeine treatment in breast, lungs and liver of DMBAinduced tumorigenesis. Cancer Research. 2022;82(12 Suppl):5850-5850.
7. Duffy MJ, Shering S, Sherry F, McDermott E, O'Higgins N. CA 15-3: A prognostic marker in breast cancer. International Journal of Biological Markers. 2000;15(4):330-333.
8. Mondal S, Bhar K, Mahapatra AS, Mukherjee J, Mondal P, Rahaman ST, et al. "Haripriya" God's favorite: Anthocephalus cadamba (Roxb.) Miq. at a glance. Pharmacognosy Research. 2020;12(1):1-7.
9. Anderson SA, Bartow BB, Harada S, Siegal GP, Wei S, Dal Zotto VL, et al. p53 protein expression patterns associated with TP53 mutations in breast carcinoma. Breast Cancer Research and Treatment. 2024;207(1):213-222.
10. Amin A. Protective effect of green algae against 7,12dimethylbenzanthracene (DMBA)-induced breast cancer in rats. International Journal of Cancer Research. 2009;5(1):12-24.
11. Kim JY, Park K, Jung HH, Lee E, Cho EY, Lee KH, et al. Association between mutation and expression of TP53 as a potential prognostic marker of triple-negative breast cancer. Cancer Research and Treatment. 2016;48(4):1338-1350.
12. Pugalendhi P, Manoharan S, Baskaran N. Effects of genistein and daidzein in combination on immunoexpression pattern of ER, PR and HER-2/neu during 7,12-dimethylbenz(a)anthracene induced mammary carcinogenesis in rats. International Journal of Research in Pharmaceutical Sciences. 2010;1(4):421427.
13. Kumar PR, Sapavatu SN, Reddy BM, Kondapalli N, Sama V, Alvi B, et al. In-silico studies on selected phytoconstituents of Neolamarckia cadamba (Roxb.) stem bark for anticancer activity. Journal of Pharmaceutical Sciences and Research. 2020;12(9):1208-1214.
14. Avila MA, Velasco JA, Cansado J, Notario V. Quercetin mediates the down-regulation of mutant p53 in the human breast cancer cell line MDA-MB468. Cancer Research. 1994;54(9):2424-2428.
15. Yadav JP, Verma A, Pathak P, Kumar V, Patel DK. Wound healing, antidiabetic and antioxidant activity of Neolamarckia cadamba, quercetin-rich extract. Pharmacological Research-Modern Chinese Medicine. 2024;11:100417.
16. Sandri MT, Salvatici M, Botteri E, Passerini R, Zorzino L, Rotmensz N, et al. Prognostic role of CA15-3 in 7942 patients with operable breast cancer. Breast Cancer Research and Treatment. 2012;132:317-326.
17. Anmar R, Omar F, Omar S, Ahmed Z, Hussein A. Diagnostic and differential efficacy of cyclin D1 and CA15-3 in breast cancer and benign breast tumors. Integrative Biomedical Research. 2023;7(1):1-6.
18. Dharmendra V, Palanivelu S, Panchanadham S. Immunomodulatory effect of Kalpaamruthaa on 7,12dimethylbenz(a)anthracene-induced mammary carcinoma studied in rats. Comparative Clinical Pathology. 2014;23(4):1087-1094.
19. Pola RK, Sapavatu SN, Bommineni MR. In silico evaluation of phytocompounds of Neolamarckia cadamba (Roxb.) stem bark for anticancer activity. International Journal of Pharmaceutical Investigation. 2021;11(3):306-312.