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. 2023 Jun 6;12(4):544–550. doi: 10.1093/toxres/tfad041

Gallic acid: a polyphenolic compound potentiates the therapeutic efficacy of cisplatin in human breast cancer cells

S Shruthi 1, Kamalesh D Mumbrekar 2, B S Satish Rao 3,4, Bhasker K Shenoy 5,✉
PMCID: PMC10470337  PMID: 37663803

Abstract

Gallic acid (GA) is a natural polyhydroxyphenolic compound with antioxidant, antimutagenic, anti-inflammatory, and antineoplastic activities. Cisplatin (CPT) is a platinum-based chemotherapeutic drug, and it is the treatment of choice for breast, ovarian, testicular, head, and neck cancers. However, the use of anticancer drugs has undesirable effects on patients due to associated toxicities. Thus, it is necessary to search for alternatives that reduce unintended side effects and enhance anticancer potential. The use of natural compounds with the conventional chemotherapeutic drug is a new aspect of cancer therapy. In the present study, we evaluated the ability of GA in the modulation of anticancer effects of CPT in human breast adenocarcinoma cells (MCF-7) by performing MTT, apoptosis, clonogenic cell survival, and micronucleus assays. GA and CPT showed significant cytotoxic activities in MCF-7 cells in a dose-dependent manner. In combination therapy (GA 2.5, 5.0, and 10 μg/mL + CPT10 μg/mL), GA synergistically reduced the MCF-7 cell viability in contrast to the individual therapies. Cancer cells death by GA is through the induction of apoptosis as observed in the acridine orange and ethidium bromide dual staining method. The frequency of micronuclei (MN) was decreased significantly (P < 0.001) in combinational therapy, possibly reducing the risk of chemotherapy-induced MN. Moreover, GA in mono or combinational therapy did not induce any cytotoxic effects in normal breast epithelial cells (MCF-10A). GA did not show any significant difference in colony inhibition compared to CPT. This outcome shows its differential effects in normal and cancerous cells. Hence, the combination GA with chemotherapeutic drugs could represent a promising alternative therapy in cancer treatment with minimal side effects.

Keywords: apoptosis, cisplatin, cytotoxicity, gallic acid, MCF-7, micronucleus

Background

Cancer is emerging as a major public health problem and is one of the leading causes of death worldwide. To minimize the current cancer burden, drug discovery is chiefly focused on the development of safe and highly effective medications with reduced unintended pharmacological effects.1 Cisplatin (CPT) is a highly recommended platinum-based chemotherapeutic drug for the treatment of patients with lung, ovarian, bladder, breast, testicular, head, and neck cancers. Cytotoxicity of CPT is due to its ability to form DNA adducts, including DNA-protein cross-links, DNA monoadducts, and inter-/intracross-links.2 The combination of CPT with other platinum-based drugs, like carboplatin, nedaplatin and oxaliplatin, is applied as a novel therapeutic strategy. This combination therapy may produce synergistic or additive effects in killing cancer cells without producing undesirable side effects.3 MCF-7 is a primary invasive breast ductal carcinoma and is frequently used in the pharmacological field to study the apoptogenic/antiproliferative properties of plant extracts on cancer cells.4–8 To determine the safety level of the test compound in human subjects, healthy cells, MCF-10A is used.9

The use of natural compounds with the conventional chemotherapeutic drug could be a promising approach for the improvement of cancer therapy. Gallic acid (GA) (C6H2(OH)3COOH) is one of the major triphenolic compounds present in plants and is considered as a bioactive nonnutritional compound due to its phytotherapeutic and antioxidant functions. It is known for its anti-inflammatory, antioxidant, free radical scavenging, and radioprotective activities.10 Our previous study has demonstrated the immunostimulating potential of GA against chemotherapeutic drugs, such as cyclophosphamide and CPT-induced immunosuppression in Swiss albino mice.11 Moreover, GA has offered protection against the chromosomal damage induced by CPT and cyclophosphamide in the bone marrow and peripheral blood cells of mice.12,13 Also, GA protected cyclophosphamide-induced reactive oxygen species (ROS)-mediated hepatic damage in mice.13 The protection offered by GA is due to its free radical scavenging ability. Abd-Rabou and coworkers14 evidenced the antitumor activity of quercetin, GA, and ellagic acid in hepatocarcinoma and colorectal cancer cell lines. Based on these findings, the present study was conducted to evaluate the possible chemopotentiation effects of GA in combination with CPT in human normal breast epithelial cells.

Materials and methods

Chemicals and reagents

GA (Lot No: MKBP6646V), CPT (Lot No.: MKBN7276V), acridine orange (AO), and ethidium bromide (EtBr) were procured from Sigma Life Science (United States). Dulbecco’s modified Eagle’s medium (DMEM), MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide), dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), trypsin, and gentamycin were obtained from Hi-Media, India.

Cell line and cell culture conditions

MCF-7 and MCF-10A cell lines were procured from the National Centre for Cell Sciences (Pune, India), and the stock cultures were preserved in liquid nitrogen. MCF-7 and MCF-10A cells were grown in T-25 flask containing DMEM supplemented with 10% FBS, 0.15% sodium bicarbonate, 2.5 mL of antibiotic-antimycotic solution, and 2 mL of gentamycin (80 μg/mL). Cultured cells were incubated in 5% CO2 with 95% humidified air at 37 °C (NuAire, Plymouth, MN, United States).

Cell counting

Cell counting was performed prior to the experimental uses to screen cells for viability.15 Briefly, cell suspension and trypan blue dye were mixed in 1:1 ratio, and the viable and dead cells were counted using the Neubauer chamber. The cell viability was calculated using the following formula:

graphic file with name DmEquation1.gif

Preparation of GA and CPT solution

GA and CPT were dissolved in double distilled water and phosphate-buffered saline (PBS), respectively, to prepare the stock solution of 1 mg/mL, and further dilutions were made as per requirement.

Cytotoxic assessment using MTT assay

The cytotoxic potential of CPT and GA was quantified using the MTT assay as per the method described by Mosmann.16 Concisely, 5 × 103 (100 𝜇L/well) were seeded in 96-well plates and were incubated in a humidified atmosphere (5% CO2 and 95% air) at 37 °C for 24 h.

MTT assay was done for following groups: group I (control): This group of culture was not treated with GA or CPT; group II (GA alone): The cells of this group were treated with different concentrations of GA (0.5, 1.0, 2.5, 5.0, and 10 μg/mL) for 24 h; group III (CPT alone): The cultures of this group were treated with different concentrations of CPT (1.0, 2.5, 5.0, 10, and 20 μg/mL) for 24 h; and group IV: (CPT + GA): The cultures of this group were treated with the IC50 value of CPT (10 μg/mL) in combination with different concentrations of GA (0.5, 1.0, 2.5, 5.0, and 10 μg/mL) for 24 h.

After 24 h of the treatment, cells were washed with PBS and were incubated at 37 °C for 4 h with 20 𝜇L (1 mg/mL) of MTT. The formazon crystals formed were dissolved in DMSO (100 𝜇L), and the absorbance was measured at 570 nm using a multiwell spectrophotometer (Tecan, Austria). Each treatment was completed in triplicates and cell viability was calculated using the following formula:

graphic file with name DmEquation2.gif

The IC50 value was determined from the concentration versus percent cell viability curve calculated using GraphPad Prism 5 (GraphPad Software, Inc., CA, United States).

Apoptosis assay

Apoptosis assay was employed to elucidate the mechanism involved in the cytotoxic effects of GA on MCF-7 cells. The morphological changes induced by GA and CPT on MCF-7 and MCF-10A cells were measured by using AO/EtBr double staining assay as per the method of Renvoizé et al.17 For the apoptotic assay, exponentially growing cells were seeded in 6-cm plates at the density of 106/plate and were incubated for 24 h at 37 °C in a 5% CO2 atmosphere. After incubation, the media was aspirated and the cells were treated with IC50 of CPT (10 μg/mL), and the effective concentration of GA (5 μg/mL) was obtained by MTT assay. After 24 h of incubation, cells were collected by centrifugation at 1,000 rpm for 5 min. Collected cells were washed with PBS and were stained with AO/EtBr (1:1 v/v) dye. Stained cells were examined under a fluorescence microscope at 400× magnification and were classified as live (L), early apoptotic (EA), late apoptotic (LA), and necrotic cells (N) as per the method of Aithal et al.18

Clonogenic assay

Clonogenic assay was performed according to Puck and Marcus.19 Clonogenic cell survival assay was used to measure the ability of cells to retain their reproductive integrity over a prolonged period time. The cells from the stock culture were trypsinized by a mild 2-min exposure to trypsin EDTA and were counted using a hematocytometer. The experiment was conducted by batch-seeding the required number of cells into 6 cm2 Petri dishes in triplicates containing 5 mL of growth medium. The optimum concentration of the compound GA (10 μg/mL) was selected from the experiment.

Group I (control): This group of culture were not treated with GA or CPT

Group II (GA alone): The cells of this group were treated with the optimum concentration of GA 10 μg/mL

Group III (CPT alone): This group of culture treated with 0.5 μg/mL of CPT

Group IV: (CPT + GA): The cultures of this group were treated concurrently with CPT (0.5 μg/mL) and GA (2.5, 5.0, and 10 μg/mL)

Following the treatment time point of 24 h, the cytotoxic agent-containing media was discarded and fresh media was added and the cultures were left undisturbed for 14 days at 37 °C in a 5% CO₂ incubator for colony formation. After the incubation period culture fixation, the media was discarded and the Petri dishes were rinsed in PBS and were stained with crystal violet, and colonies containing ≥50 cells were counted. The plating efficiency (PE) and survival fraction (SF) were calculated as:

graphic file with name DmEquation3.gif
graphic file with name DmEquation4.gif

Micronucleus assay

The MN test was performed to detect the genetic damage induced by GA and CPT, and it was performed as per the method described by Das et al.20 Cells were divided into the following groups: group I (control): This group of culture was not treated with GA or CPT; group II (GA alone): The cells of this group were treated with the optimum concentration of GA, 10 μg/mL; group III (CPT alone): This group of cultures was treated with 10 μg/mL of CPT; and group IV: (CPT + GA): The cultures of this group were treated concurrently with CPT (10 μg/mL) and GA (2.5, 5.0, and 10 μg/mL) for 24 h. On posttreatment, cytokinesis was blocked by treating the cells with cytochalasin-B (3 μg/mL) and was further incubated for 24 h to obtain binucleated cells. Cells were harvested by trypsinization and were rinsed with PBS and centrifuged at 1,553 g for 10 min to obtain the pellet. The pellets were resuspended in hypotonic solution (0.56% KCl) for 3 min to ensure cytoplasmic bulging followed by fixing with ice-cold Carnoy’s fixative (methanol:acetic acid, 3:1). Next, the fixed cells were smeared on clean glass slides, air dried, and stained with AO (20 mg/mL, prepared in Sorensen’s phosphate buffer, pH 6.8) and were then scored under a fluorescent microscope (Olympus BX51, Olympus Microscopes, Tokyo, Japan). One thousand binucleated cells with intact cytoplasm, which stain red, and nucleus/micronucleus, that stain green, were scored, and the number of micronucleated binucleate cells was noted from 3 independent experiments.

Statistical analysis

The experimental data were expressed as mean ± SEM. The statistical significance of the data was analyzed by employing 1-way ANOVA and Dunnett’s post hoc tests using GraphPad Prism 5 (GraphPad Software, Inc., CA, United States). A P-value of ≤0.05 was considered to be statistically significant.

Results

Effect of GA and CPT on MCF-7 and MCF-10A cells’ viability

In treated cells, a dose-dependent reduction in the viability of human breast adenocarcinoma cells at all treatment regimens was observed. The dose of CPT required for 50% growth inhibition of MCF-7 cells was found to be 10 μg/mL for 24 h. In combined treatment groups, a maximum cellular death of 87.3% was seen in GA-treated (5 μg/mL) + CPT-treated (10 μg/mL) cells (Fig. 1A). A significant elevation of cell survival (21.5%–47.2%) was observed in GA-treated (0.5, 1.0, 2.5, 5.0, and 10 μg/mL) cells in comparison with control untreated cells. MCF-10A cells treated with CPT (10 μg/mL) showed 51.8% of cell death, indicating its cytotoxicity on normal cells. In the combinational treatment group, a maximum of 40.6% enhancement in cell survival was observed at GA 0.5 μg/mL when compared to CPT-alone-treated cells (Fig. 1B).

Fig. 1.

Fig. 1

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Percentage of viable cells treated with GA and CPT at 24-h exposure. A) MCF-7 cells; B) MCF-10A cells; Values are mean ± SEM, (n = 3), 1-way ANOVA followed by Dunnett’s post hoc test; *P < 0.05 compared to control cells; **P < 0.01 compared to control cells; ***P < 0.001 compared to control cells; #P < 0.05 compared to CPT; ##P < 0.01 compared to CPT; ###P < 0.001 compared to CPT.

Cell death induced by CPT and GA on MCF-7 and MCF-10A cells

The nuclei and plasma membrane of untreated control cells were found to be intact and were stained with AO and appeared green in color (Fig. 2A). CPT-treated MCF-7 cells showed the presence of N cells in which EtBr penetrated the membranes of dead cells and stained their nuclei, red in color (Fig. 2B). The EA and LA cells were observed in MCF-7 cells treated with GA (5 µg/mL)+CPT (10 µg/mL). EA cells appeared bright green in colour with condensed nuclei and LA cells with condensed and fragmented nuclei, stained orange in colour  (Fig. 2C).

Fig. 2.

Fig. 2

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GA and CPT induced nuclear morphological alterations in MCF-7 cells assessed using AO/EtBr staining. A) Untreated control cells showing L cells with normal round nuclei stained green; B) CPT-treated (10 μg/mL) cells showing N cells (stained red); C) GA-treated (5 μg/mL) + CPT-treated (10 μg/mL) cells showing bright green and orange apoptotic cells with condensed and/or fragmented nuclei; magnification, 400×.

Treatment of cells with GA (5 μg/mL) showed significant (P < 0.01) decrease in the percentage of viable MCF-7 cells. The percentage of early and LA cells was found to be more in GA (5 μg/mL) and CPT (10 μg/mL) combined treated cells compared to CPT-treated (10 μg/mL) cells (Fig. 3A). Most of the cells treated with GA (5 μg/mL) have undergone cell death through the apoptotic pathway rather than the N pathway. The protective effects of GA against CPT-induced apoptosis and necrosis in MCF-10A cells are illustrated in Fig. 3B. MCF-10A cells treated with GA (5 μg/mL) did not show any nuclei morphological alteration compared to untreated control cells (P < 0.001). MCF-10A cells treated with CPT revealed the presence of more EA, LA, and N cells when compared to untreated control MCF-10A cells (P < 0.001). GA (5 μg/mL) treatment enhanced the MCF-10A cell viability compared to untreated control cells (P < 0.001); also, it did not induce any effect on the survival of normal cells. GA (5 μg/mL) in combination with CPT (10 μg/mL) showed a reduction in the percentage of EA, LA, and N cells compared to CPT-alone-treated (P < 0.001) cells.

Fig. 3.

Fig. 3

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Percentage of viable, apoptotic, and N cells treated with GA and CPT at 24 h. A) MCF-7 cells; B) MCF-10A cells; Values are mean ± SEM, (n = 3), 100 cells scored from each treatment group. One-way ANOVA followed by Dunnett’s post hoc test; P < 0.05 compared to control cells; **P < 0.01 compared to control cells; ***P < 0.001 compared to control cells; #P < 0.05 compared to CPT; ##P < 0.01 compared to CPT; ###P < 0.001 compared to CPT.

Clonogenic assay

Clonogenic assay was used to assess the reproductive death of MCF-7 cells after treatment with GA alone and in combination with CPT. GA did not show significant cell death compared to CPT-treated cells. However, the effect produced by GA + CPT is found to be significant when compared with the control untreated cells. Among the 3 doses of GA tested, 10 μg/mL showed maximum reproductive cell death in combination with CPT (Fig. 4).

Fig. 4.

Fig. 4

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A) Colony formation of MCF-7 cells after exposure to GA alone and in combination with CPT. B) Surviving fraction of MCF-7 cells treated with GA and CPT. Mean ± SEM (n = 3), *P < 0.001 compared to control cells; **P < 0.001 compared to control cells; NS compared to CPT.

Micronucleus assay

GA did not induce MN in MCF-7 cells following 24-h treatment. In combinational therapy, a significant decrease in the frequency of MN was observed at all the 3 doses of GA tested as compared with CPT-alone-treated MCF-7 cells. GA-treated (10 μg/mL) + CPT-treated (10 μg/mL) cells showed 52% (P < 0.001) decline in the frequency of MN when compared to the CPT-alone-treated group (Fig. 5). The maximum MN reduction (P < 0.001) was observed at GA 10 μg/mL.

Fig. 5.

Fig. 5

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Effect of GA and CPT on the MN induction in MCF-7 cells. MN were scored in 1,000 BN cells; mean ± SEM (n = 3), **P < 0.001 compared to control cells; #P < 0.05 compared to CPT; ##P < 0.001 compared to CPT.

Discussion

Breast cancer is the most common type of cancer, causing mortality and morbidity among women worldwide.7 CPT is a well-known anticancer agent with a high rate of success in treating a wide variety of cancer, namely lung, neck, cervical, bladder, and ovarian.21 CPT induces cell death in cancer cells through the apoptosis mechanism and is manifested by distinct morphological changes in the cell. Administrating CPT in combination with other drugs, like paclitaxel, capecitabine, and Chinese herbal medicine, is one of the strategies to overcome CPT resistance and toxicity.22 Improvement of the overall outcome of CPT in chemotherapy is achieved by treating the patients with compounds enhancing the effect of apoptosis.23 In search of newer natural therapies for cancer prevention, cancer cell lines have been widely used in research. Cancer cell lines are proven to be an appropriate tool in the genetic approach and are an excellent model for the study of the biological mechanisms involved in cancer.24 In fact, for the study of proliferation, deregulation, apoptosis, and cancer progression, the use of the appropriate in vitro model in cancer research is crucial.25 Earlier evidence indicates that CPT is used in combination with natural compounds like osthole in lung cancer cell lines, vinblastine and bleomycin in metastatic granulosa cell tumor of the ovary, honey bee venom in ovarian cancer cells, and anvirzel in breast, colon, lung, prostate and melanoma.3 GA is a plant-derived phenolic compound, and it has been reported to prevent the development and progression of oral, lung, and pancreatic cancers through the regulation of apoptosis.26 The current study aimed to investigate the potentiating effects GA on the chemotherapeutic effects of CPT in MCF-7 cells.

The pure compounds with an IC50 value <4 μg/mL on cell lines were considered to be potent cytotoxic compounds. The preclinical evaluation of such compounds using animal models is carried to check their chemotherapeutic potential.27 The IC50 value of GA on MCF-7 cell lines at 24 h was found to be 20 μg/mL (117.6 μM). Hence, in our study, rather than assessing its individual cytotoxic effect, the modulatory effect of GA on clinically utilized chemotherapeutic drug CPT was evaluated. Accordingly, combinational cytotoxic effect of GA (0.5, 1.0, 2.5, 5.0, and 10 μg/mL) with CPT against MCF-7 was evaluated. In MTT assay, as expected, after 24 h of incubation, CPT inhibited MCF-7 cells growth in a dose-dependent manner. The IC50 value of CPT in MCF-7 cells was measured at 24 h and was found to be 10 μg/mL (33.21 μM), similar to reported earlier.28 A decrease in cell viability was noticed in MCF-7 cells following 24 h of GA exposure. Meanwhile, GA in combination with CPT has potentiated the inhibitory effect of CPT on cancer cells in a dose-dependent manner. The maximum MCF-7 cell inhibition (87.3%) was observed at GA 5 μg/mL in combination with CPT (10 μg/mL). Interestingly, GA has shown its distinctive mode of action on cancerous and normal cells. As evident from MTT results, CPT has shown its cytotoxicity not only on cancer cell lines but also on normal cells. By contrast, combining GA with CPT has offered protection against the toxicity of CPT in normal cells. The percentage of viable cells was observed to be more in the combined treatment group in comparison with the CPT-alone treatment. This observation strongly suggests the cytotoxic activity of GA in cancer cells without causing damage to normal cells. Based on the data obtained, cytotoxicity of GA is more selective to MCF-7 cells rather than MCF-10A cell lines. Whereas, MCF-10A cells treated with GA alone showed an increased proliferation rate and viability. This chemotherapeutic potentiating effect of GA in combination with CPT is in agreement with the study by Aborehab et al.,29 where they show that the major mechanism involved in cell death is the ability of GA to induce cell cycle arrest followed by apoptosis.

The AO/EtBr dual staining results confirmed that the cytotoxicity of GA toward MCF-7 cells is induced by apoptosis. MCF-7 and MCF-10A cells treated with CPT (10 μg/mL) showed an upsurge in the percentage of apoptotic and N cells compared with the untreated control cells. Furthermore, the test revealed the presence of cells with membrane blebbing, chromatin condensation, and fragmentation following 24-h treatment with CPT. Aithal et al.18 suggest that CPT-induced apoptosis or necrosis is due to the overload of intracellular ROS. CPT is an alkylating agent, and hence, this kind of anticancer drug induces apoptosis/necrosis through oxidative stress-mediated mechanism. As evidenced, GA treatment augmented the cytotoxic effect of CPT on MCF-7 cells with the significant increase in early and LA cells when compared to CPT-alone-treated cells (P < 0.001). The reduction in the number of viable MCF-7 cells and the presence of morphological apoptotic changes in cells treated with GA in combination with CPT indicate their cytotoxic and apoptotic potential on MCF-7 cells. Cell death induced by GA followed the sequence of early and LA morphological changes; representing cell death induced by GA is through an apoptotic pathway rather than N. Importantly, GA was found to be more selective for cancer cells and less toxic than CPT. GA conferred protection against CPT-induced cellular death on the MCF-10A cells. Similar lines of evidence indicate that the GA-induced apoptotic effects were due to the triggering of the extrinsic Fas/Fas L pathway as well as the intrinsic or mitochondrial pathway.30

GA is a known polyphenolic compound, and there are few reports available on the cytotoxic effects of polyphenols on cancerous cells. In this line, Padma et al.,31 worked on the apoptotic effects of a natural polyphenol component mangiferin on rhabdomyosarcoma cancerous cells. They reported that mangiferin-induced apoptosis was due to the sustained generation of oxidative stress in rhabdomyosarcoma cells. Oxidative stress-mediated apoptosis induced by Flueggea leucopyrus extract on human endometrial carcinoma (AN3CA) cells have been reported by Samarakoon et al.32 Behzad et al.33 showed the cytotoxic and apoptotic potential of Primula auriculata extract in HT-29 human colon adenocarcinoma cells. The presence of phenolic compounds like flavonoids and saponins may be attributed to its cytotoxic and apoptotic effects. Based on the reports available on the cytotoxic effects of polyphenols and flavonoids, the possible cytotoxic and apoptotic effect of polyphenol GA is due to the induction of enhanced oxidative stress in cancerous cells.

The colony forming ability of GA alone and CPT combination treated MCF-7 cells was evaluated using the colony forming assay. The survival fraction of MCF-7 cells obtained in GA treated group is not significantly different from the value obtained with the control. In contrast, significantly lower survival fraction was observed in combined treatment (GA+CPT) groups than in control.GA alone or in combination did not show significant reproductive cell death when compared with CPT. While, as observed from the results of MTT assay GA has synergistically enhanced the cytotoxicity of CPT on cancer cells with relatively high selectivity. The observations made by Wang et al.34 showed the synergistic effect of GA on the enhancement of the anticancer effects of CPT in human small cell lung cancer H446 cell line via the ROS-dependent mitochondrial apoptotic pathway.

MN assay results show that the combination of GA and CPT did not induce more micronuclei (MN) than CPT alone. The therapeutic effect of CPT is based on its ability to induce DNA adducts, which ultimately leads to the blocking of DNA replication.2 The MTT results of the present study indicated that the combination of GA and CPT has synergistic interactions, predominantly observed as increased cell death. However, GA did not potentiate the level of CPT-induced chromosomal aberrations but significantly reduced cancer cell survival by inducing apoptosis as noticed by MN and apoptosis assays. Chromosomal damage can be considered as a biomarker for cancer risk.35 The results discussed above demonstrated that GA reduced the risk of CPT-induced MN in cancer cells, which may be due to the overkill effects. The elimination of highly damaged cells from a pool of cells reduces the frequency of chromosomal damage in surviving cells.36

Conclusion

In conclusion, the study showed that GA has the potential to exert the cytotoxic effects of CPT on human breast adenocarcinoma cells. Cytotoxicity of GA on cancer cells is through the induction of apoptosis and by reducing the risk of chromosomal damage. Hence, the combination of CPT and GA could represent a promising alternative therapy for breast cancer treatment with reduced doses and minimal side effects. Further studies on understanding the underlying mechanism in the interaction of GA with CPT may provide a valuable insight into cancer therapy.

Acknowledgments

The guidance and encouragement of Late Professor K. K. Vijayalaxmi are gratefully acknowledged.

Contributor Information

S Shruthi, Department of Postgraduate Studies in Applied Zoology, Alva’s College, Vidyagiri, Moodbidri, Dakshina Kannada, Karnataka 574227, India.

Kamalesh D Mumbrekar, Department of Radiation Biology and Toxicology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka 576104, India.

B S Satish Rao, Department of Radiation Biology and Toxicology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka 576104, India; Research Directorate Office, Manipal Academy of Higher Education, Manipal, Karnataka 576104, India.

Bhasker K Shenoy, Department of Applied Zoology, Mangalore University, Mangalagangothri, Dakshina Kannada, Karnataka 574199, India.

Authors’ contributions

K. Bhasker Shenoy and B.S. Satish Rao, conceptualized and supervised the study. S. Shruthi conducted research and drafted the manuscript. S. Shruthi and Kamalesh D. Mumbrekar analyzed the results. All authors revised and approved the final manuscript.

Conflict of interest statement: The authors declare that there is no conflict of interest.

Data availability

All the data are contained in the manuscript.

Consent for publication

Not applicable.

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