Effect of turmeric extract (Curcuma longa) on apoptosis in a rat model of mammary adenocarcinoma with adriamycin-cyclophosphamide chemotherapy

Breast cancer is now the most commonly diagnosed cancer in the world. A considerable number of cases are diagnosed annually that indicating it as a leading life-risk disease in women worldwide. Over 2.3 million new cases are annually detected globally, according to the report of GLOBOCAN 2020 [1]. Breast cancer ranks as the most prevalent cancer in Indonesia, with an incidence of 42.1 per 100,000 population ( per 100,000 population) and a mortality rate of 17 per 100,000 population [2]. Common risk factors for breast cancer include biological sex, family history, presence of genetic factors, and lifestyle behaviors such as obesity, alcoholism, and physical inactivity [3].

Adriamycin (doxorubicin) is a well-characterized inhibitor of tumor growth and has been used for many years as a combined therapy for cancer. Adriamycin is selected due to its unique mechanism to damage DNA through intercalation into the DNA strand and also by inhibition of topoisomerase II which induces in cell death [4, 5]. The uses of adriamycin is a dose and time dependent manner. However, high dose msay induce side effects and comparably low dose with short duration can be used. Overall response rate to doxorubicin-based chemotherapy in triple-negative breast cancer (TNBC) has been varying between 40% and 70% [2, 3]. The differences in response rates are in part because there are no biomarkers to identify patients who will respond before treatment [5].

The approach of breast cancer control varies due to clinical intervention to multimodality treatment, involving local and systemic therapies, e.g., surgery and/or chemotherapy [6]. Several common chemotherapies are used in the treatment of breast cancer including combination of damage-inducing cytotoxic drugs such as Adriamycin and Cyclophosphamide (AC), which work by targeting tumor cells DNA and it lead to inhibit cancer-cell growth. Chemotherapy is effective but often induces significant side effects, including fatigue, nausea, fertility issues, and risks of cardiomyopathy and drug resistance [7-9].

Traditional herbal therapeutics are getting more attention as an alternative treatment of cancer care [10]. Turmeric (Curcuma longa) has multiple health benefits, used as an anticancer potential due to contain of active ingredients [4]. Curcumin has also been used for a variety of other biological effects, such as anti-inflammatory and anti-oxidative effects. The antitumor activity of curcumin is mainly mediated through apoptosis induction, tumor proliferation, and invasion inhibition [11].

Caspase-3 is the main executor of apoptosis in human patients, and the expression level of caspase-3 is often linked to cancer cell death induction. In many studies, curcumin was efficiently involved in stimulating caspase-3 activity, inducing the death of cancer cells [12]. Studies on lung cancer, prostate cancer, and several others have indicated that curcumin is capable of suppressing cancer cell growth in this manner [13]. Still, there are a few areas that have not been well explored. For example, the role of turmeric in breast cancer, particularly its potential in caspase-3 expression and apoptosis index [14].

More research is required to categorize the role of turmeric to improve apoptosis in breast cancer, including in combination with chemotherapy. Thus, the present study primarily aims to evaluate the benefits of turmeric extract on caspase-3 expression and apoptosis index in mammary adenocarcinoma to identify new strategies for breast cancer therapy with bioactive components that can be effective with minimal side effects.

Ethics statements

The health research ethics committee of the Faculty of Medicine Diponegoro University (Protocol Number: 064/EC-H/KEPK/FK-UNDIP/VII/2024) approved this study that was carried out according to the ethical principles of animal research respecting 3R (Replacement, Reduction, Refinement) as well as 5F (Freedom from hunger and thirst, discomfort, pain, injury or disease, fear and distress, and freedom to express normal behavior).

Population and research sample

The study population of 5 weeks old-Balb/c female mice with the inclusion criteria involved mice that had been successfully inoculated with mammary adenocarcinoma with body weight between 100-150 grams and were in good health. The anatomical defects or mice showing illness and inactive behavior during induction or treatment were not selected for the experiment. The normal mice were considered for this study. The study used 24 mice and divided into four groups with each group having six mice randomly assigned. One extra mouse was added in each group in case of dropouts. The samples were randomly selected, and sample size calculations were based on WHO guidelines and the Institutional Animal Care and Use Committee Guidebook.

Sample preparation

The study was carried out over a 3-month window. The turmeric extract was prepared in the Stem Cell and Cancer Research Laboratory at Gadjah Mada University in Yogyakarta, and treatments and tissue sampling of the mice were performed in this Lab. Turmeric extract was indeed the independent variable in this study, whereas caspase-3 protein expression and the apoptosis index were studied as dependent variables.

Animal study

The mouse groups were treated and acclimatized for 1 week with standard feed. The treatment groups were included as: K+ (100 mg/kg BD Adriamycin-cyclophosphamide), P1 (100 mg/kg BD Adriamycin-cyclophosphamide followed by Turmeric extract at the dose of 100 mg/kg b.w.), P2 (100 mg/kg BD Adriamycin-cyclophosphamide followed by Turmeric extract at the dose of 150 mg/kg/b.w.), and P3 (100 mg/kg BD Adriamycin-cyclophosphamide followed by Turmeric extract at the dose of 200 mg/kg b.w.).

For tumor induction, the mice were administered with DMBA (7,12-dimethylbenz[a]anthracene) from Sigma-Aldrich, St. Louis, United States, at a dose of 20 mg/kg body weight through subcutaneous injection twice a week for 5 weeks. Evaluation of tumor nodules in the mammary glands was performed weekly for 2 weeks post-DMBA induction. After establishing the implanted tumor model, the mice were classified into multiple treatment groups and treated with 5 weeks of chemotherapy with Adriamycin-Cyclophosphamide along with vitamins according to specific doses for each group.

Caspase-3 protein expression analysis

Caspase-3 protein expression was studied by immunohistochemistry from Invitrogen, Waltham, United States using rat polyclonal primary antibody against caspase-3. Tumor cells were stained brown and the cytoplasm and tumor cells in 10 high-power fields (400x magnification) were investigated to assess the tumor cell index. The apoptosis index was evaluated by the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) technique using a tool from VitroVivo Biotech, Rockville, United States, which estimates the number of apoptotic cells in 10 fields at high-power magnification (400x) among tumor cells.

Statistical analysis

Data for caspase-3 protein expression and apoptosis index were analysed using SPSS software (version 21.0). Data are described as means, standard deviations, medians, and boxplots (for nonnormally distributed data). The Shapiro-Wilk test was used to conduct normality tests. Assuming normality, a one-way ANOVA followed up by a Post Hoc Bonferroni test was conducted. For non-normally distributed data, a non-parametric Kruskal-Wallis test and a Mann-Whitney test were applied. All data were considered significant at p < 0.05.

Effect of turmeric extract on Caspase-3 expression

The effect of turmeric extract on Caspase-3 expression in tumor cells was observed using a microscope (Figure 1).  A variation of cellular responses was observed among the treated groups compared to the control. The treatment groups were statistically significant (Table 1). The differences in Caspase-3 expression were also observed between the K+ group and the P1 and P2 groups (Table 2).

Table 1. Caspase-3 descriptive and normality test results.

Table 2. Differences in levels of Caspase-3 between treatments.

Effect of turmeric extract on apoptosis index

Turmeric extract influences the apoptosis index. Following treatment in tumor cells, a significant variation was observed using a microscope (Figure 2). The apoptosis index showed significant differences among K+, P1, P2, and P3 groups (Table 3). However, a highly significant difference in apoptosis index was observed in the P1 and P2 groups as compared with the K+ group (Table 4).

Table 3. Apoptosis Index descriptive and normality test results.

Table 4. The apoptosis index difference test between treatments results.

Breast cancer is one of the most prevalent cancers and a leading cause of mortality among women worldwide. The high incidence of this disease is often attributed to various risk factors, including age, genetic predisposition, environmental influences, obesity, and alcohol consumption, particularly in developing countries [3]. Traditional treatments for breast cancer, such as surgery, radiotherapy, and chemotherapy, are effective but often come with significant adverse effects and can lead to the development of chemotherapy resistance, which remains a major challenge in oncology. Consequently, complementary therapies, including herbal medicine, are gaining recognition as potential alternatives, with turmeric (Curcuma longa) being a notable example [6].

Turmeric, widely used in South Asia, Indonesia, and India, contains a high concentration of curcumin, a compound known for its anticancer effects [10]. The primary molecular mechanisms through which curcumin exerts its anticancer effects include the induction of apoptosis, inhibition of cell proliferation, and disruption of tumor invasion processes. Research indicates that curcumin is safe at specific doses and can target a variety of cancer cells, including breast cancer cells [15].

Our findings indicate that the expression of caspase-3 was significantly higher in the group treated with curcumin compared to the control group. This increase in caspase-3 expression aligns with the study by Bhattacharya et al., which demonstrated that curcumin can modulate caspase-3 expression and induce apoptosis in cancer cells [16]. Curcumin, as a natural polyphenol, exhibits broad-spectrum anticancer activity through its antiproliferative, antioxidant, anti-inflammatory, and pro-apoptotic properties [4].

Curcumin has been shown to interfere with critical signaling pathways involved in cancer pathogenesis, such as the PI3K/Akt and NF-κB pathways. Additionally, curcumin plays a significant role in reducing HER2 expression in breast cancer, which enhances the regulation of apoptosis-related proteins, including caspase-3 and caspase-9, while inhibiting the anti-apoptotic protein Bcl-2. The overexpression of caspase-3 observed in this study indicates a successful induction of apoptosis in breast cancer cells following treatment with curcumin, particularly in conjunction with Adriamycin-Cyclophosphamide [17].

The apoptosis index in the curcumin-treated groups was significantly higher than that in the control group, corroborating findings by De Jong et al., who reported a substantial impact of curcumin on the apoptosis index [18]. Apoptosis is a crucial mechanism in cancer treatment, as it can lead to tumor shrinkage and help prevent metastasis. The apoptosis index serves as an important indicator of tumor response to treatment, especially in the context of chemotherapy [19].

Apoptosis index is one of the important indicators relevant to tumor response for treatment, especially for chemotherapy [20]. In this study, curcumin was shown to modulate the expression of apoptosis-related proteins such as Bax and Bcl-2, thereby enhancing the propensity of cancer cells to undergo apoptosis [21]. This mechanism suggests that curcumin not only aids in reducing tumor cell growth but also enhances the effectiveness of conventional treatments [22].

It is suggested from this study that curcumin significantly increases the expression of caspase-3 and the apoptosis index in breast cancer cells, positioning it as a promising complementary therapy in breast cancer treatment. Curcumin not only enhances the efficacy of conventional therapies but is also reported to reduce the side effects associated with chemotherapy.

This study demonstrates that turmeric extract (Curcuma longa) significantly enhances caspase-3 expression and the apoptosis index in Adriamycin-Cyclophosphamide-induced breast cancer cells, indicating its strong antitumor activity. The ability of curcumin to induce apoptosis suggests its potential to reduce tumor size, prevent metastasis, and overcome therapeutic resistance. These findings highlight curcumin as a promising adjunctive agent in breast cancer treatment, potentially enhancing chemotherapy effectiveness with minimal side effects. Further research is needed to confirm its broader therapeutic applications.

Thanks to the Department of Pathological Anatomy Gadjah Mada University for providing the facility needed to conduct the research.

All the authors designed outlines and drafted the manuscript, performed the experiments, analyzed the data, wrote the initial draft of the manuscript, and reviewed the scientific contents described in the manuscript. All authors read and approved the final submitted version of the manuscript. 

There is no conflict of interest among the authors.