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. 2019 Oct 29;9(11):428. doi: 10.1007/s13205-019-1930-2

Evaluation of the combinatorial effect of Tinospora cordifolia and Zingiber officinale on human breast cancer cells

Gitanjali Javir 1,2, Kalpana Joshi 2,✉
PMCID: PMC6820643  PMID: 31696033

Abstract

The present study was aimed to investigate the anticancer potential of the combination treatment of Tinospora cordifolia (TC) and Zingiber officinale (ZO) using network pharmacology approach. In silico analysis of the anticancer activity of TC + ZO was carried out using Cytoscape 3.2.0 software to elucidate the mechanism. The MTT assay confirms the combination of TC and ZO is more active (IC50; 2 μg ml−1) as compared to TC (509 μg ml−1) and ZO (1 mg ml−1) alone in MCF-7 cells. The TC + ZO combination treatment inhibits DNA synthesis, migration, and induces apoptosis in MCF-7 cells as compared to TC and ZO alone at a concentration of 1 µg ml−1. TC + ZO combination treatment arrested cell cycle significantly at the G0/G1 phase. The proposed synergistic activity of the two herbs in the treatment of several cancers was correlated with an appropriate associated target/s, based on the pharmacological network. Interestingly, when both the plants used in combination, were found to regulate a total of 16 genes in 27 types of cancers. Further, ALOX5, MMP2, and MMP9 genes were identified as major targets which are responsible for the TC + ZO anticancer activity. According to merged and sub-networks of source-bioactive, bioactive-target, target-disease of TC, ZO alone and their combination; MMP9 was selected for validation purpose. The real-time PCR analysis confirmed that the TC + ZO combination treatment significantly down-regulated MMP9 mRNA expression by fivefold via up-regulation of its downstream target ER-α by 3.5-fold. In conclusion, the network analysis and in vitro validation confirmed the potent synergistic activity of TC + ZO combination treatment in breast cancer.

Electronic supplementary material

The online version of this article (10.1007/s13205-019-1930-2) contains supplementary material, which is available to authorized users.

Keywords: T. cordifolia, Z. offificinale, Anticancer, MMP9, ER-α, Network pharmacology

Introduction

Breast cancer accounts for 30% of all new cancer diagnoses in US women. The most common cancers such as breast, lung, colorectum, and prostate, account for 45% deaths. An estimated 609,640 people will die due to cancer, corresponding to almost 1700 deaths per day in 2018 (Siegel et al. 2019). The selective estrogen-receptor modifier drug, tamoxifen, leads to an increased risk of resistance to cancer cells, thromboembolic complications, endometrial cancer, and endometrial hyperplasia after prolonged use (Nounou et al. 2015). Hence, there is an urgent need to develop potent therapies to treat cancer.

The natural compounds mediate anticancer effects via multiple pathways. For example, bitter melon exerts an anticancer effect in head and neck cancer via immunomodulatory (Bhattacharya et al. 2016) and natural killer cell-mediated toxic effect (Bhattacharya et al. 2017) whereas in breast cancer it inhibits cancer cell growth via induction of autophagy and modulation of AMPK/mTOR pathway (Muhammad et al. 2017). Tinospora cordifolia (TC) and Zingiber officinale (ZO) are widely used herbs in ayurvedic medicines.

Tinospora cordifolia (TC) is a divine herb commonly known as Guduchi. It is known to possess immunomodulatory, anti-inflammatory, anti-diabetic, anti-arthritic, anti-malarial, radiosensitizing (Rao et al. 2008), anti-cancer (Jagetia and Rao 2006) anti‑obesity, cardioprotective, hepatoprotective and neuroprotective, anti-bacterial (Jagetia and Rao 2006; Mishra et al. 2013) properties. It is large, climbing shrub with elongated branches and heart-shaped leaf found at a higher altitude of the Indian sub-continent and Chinese tropical area. Flowers are unisexual, greenish-yellow in color, and appear when the plant is leafless. Fruits of TC are aggregated in a cluster of one to three. It belongs to class-Angiosperms, order-Ranunculales, family-Menispermaceae, and kingdom-Plantae.

Zingiber officinale is one of the widely used spices worldwide and commonly known as Ginger. ZO is reported to have anti-inflammatory (Funk et al. 2016), anti-oxidant, anti-diabetic (Li et al. 2017a), anti-arthritic (Funk et al. 2016), anticancer, anti-neurodegenerative (Seow et al. 2017) properties. It is the flowering plant of which rhizome is used as a spice and in folk medicines. ZO is a perennial reed-like herb having a leafy stem. It grows up to 3.4 m tall belonging to class-Angiosperms, order-Zingiberales, family-Zingiberaceae, and kingdom-Plantae. Primarily it was originated from Southeast Asia and grown in Southern India. As per the survey of the Food and Agricultural Organization of the United Nations, Statistics Division in 2016, India ranked first in the production of ZO.

A combination of TC and ZO is used for the treatment of arthritis. TC and ZO formulation along with Emblica officinalis and Boswellia serrata showed potent clinical efficacy in symptomatic knee osteoarthritis patients (Chopra et al. 2013). Wherein, ZO is reported to block NF-κβ pathway and TC is used as an immune modulator. In certain diseases wherein current therapies found to be inadequate, the repurposing of drug opens up an opportunity for the therapeutic options for such diseases. For example, methotrexate a popular drug, which is used at low doses as anti-arthritic and high doses for cancer treatment, now in phase 2 of cancer clinical trial (Fiehn et al. 2004). Auranofin is another drug approved for rheumatoid arthritis but currently, it is in phase 2 clinical trial of chronic lymphocytic leukemia (Roder and Thomson 2015). Artemisinin was approved for the treatment of malaria now it has been used in the treatment of cancer, arthritis, diabetes, etc. (Yuan et al. 2017).

Literature form the last 2 decades have proven that the TC and ZO combination is effective for arthritis treatment (Chopra et al. 2011; Chopra et al. 2013). As our interest was in exploring the repurposing of TC, ZO and TC + ZO combination in cancer therapy, in the present study we have used a newer NP approach to support our hypothesis. Ayurvedic preparations have complex chemical compositions hence investigation of the pharmacological mechanism of activity remains challenging. The concept of network pharmacology (NP) has been successfully used for the investigation of traditional Chinese medicines (TCM), Korean medicines and western medicines. In the present study, we used chemo-informatics and network pharmacology approach to predict the mechanism of activity of the ayurvedic drug combination TC + ZO in several diseases. Further, we demonstrated for the first time the synergistic anticancer potential of the combination of TC + ZO using in vitro assays. To support our hypothesis we have validated our in silico findings with quantitation of lead target by mRNA expression. This study will serve as the model to study the mechanisms of ayurvedic formulations. Additionally, NP is also useful in the experimental design of widely studied entities.

Method

Preparation of extract

Tinospora cordifolia (TC) and Zingiber officinale (ZO) extracts in powder form were obtained from Pharmanza Herbal Pvt. Ltd., Mumbai, India. The 10 mg of TC and ZO powder was dissolved separately in 10 ml of distilled water. Each of them was subjected to cold maceration at 4 °C for 72 h followed by lyophilization to remove the solvent. Each of lyophilized aqueous extracts was dissolved in distilled water at concentration 1 mg ml−1 then diluted in DMEM medium and used for cell culture experiments. For the TC + ZO combination, 5 mg of TC and 5 mg of ZO was dissolved in 10 ml distilled water to obtain 10 mg/ml concentration. Further, diluted in DMEM and used for different assays. The characterization of aqueous extracts was carried out by HPLC using their major constituent as shown in Supplementary file (Figs. S2–S6). The retention of the reference standard was compared with the extract and chromatograms are as shown in Supplementary file (Figs. S2, S4). TC and ZO were dissolved in 1 mg ml−1 in distilled water, filtered through 0.22 µm filter paper. They were used for HPLC and to prepare the drug dilutions for in vitro assays.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

MCF-7 (breast cancer) cell line was procured from NCCS, Pune, India, and maintained in DMEM with 10% FBS and 1% anti-mycotic-anti-biotic solution in 5% CO2 atmosphere at 37 °C. 1 × 105 cells per ml per well were seeded in 96-well plates. Cells were incubated with different concentrations (0, 0.001, 0.01, 0.1, 1, 10, 100 μg ml−1) of aqueous extract of TC, ZO and TC + ZO for 24 h. After treatment, the medium was replaced with 20μL of 5 mg ml−1 MTT [3-(4,5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide] and cells were incubated for another 4 h at 37 °C. Further, MTT solution was removed and insoluble formazan crystals were dissolved in 100 μl of DMSO (dimethyl sulfoxide). The absorbance was read at 570 nm using a microplate reader.

BrdU incorporation assay

The 1 × 105 cells per ml were incubated with or without TC, ZO and TC + ZO extract at 1 μg ml−1 and 10 μg ml−1concentrations. After 24 h treatment, 20 μl of BrdU (1×) was incorporated into cells and incubated for another 24 h. BrdU incorporation assay was carried out using a BrdU cell proliferation kit (Cell Signaling Technology, MA, USA) as per manufacturer’s protocol.

Cell cycle analysis by flow cytometry

The 1 × 105 cells per ml were treated with TC, ZO, TC + ZO, and tamoxifen. After treatment of cells for 24 h, they were washed with 1× PBS and fixed in 70% ethanol at − 20 °C overnight. Before analysis, cells were treated with 25 μl of RNase A (20 mg ml−1), 2 mM MgCl2 and 5–10 μl of 100 µg ml−1 propidium iodide, followed by incubation for 10–15 min at room temperature. DNA contents were recorded by flow cytometer (Becton–Dickinson) followed by data analysis on Cell Quest software.

Wound scratch assay

A linear wound was created across the center of each well with a 200 μl plastic tip when cells were at a confluency ≥ 90%. The wounded monolayer was washed three times with 1× PBS to remove cell debris and incubated with TC, ZO, TC + ZO extracts and tamoxifen for 24 h. Photographs and areas of the wounds were captured at 0 h and 24 h using Axivert 40CFL Zeiss microscope. Percent of areas covered by cells were calculated by considering 0 h as 100% empty area. All the experiments were performed in triplicates.

Annexin-FITC/PI double staining

Briefly, 1 × 105 cells per ml used for the assay. Annexin-FITC/PI dual staining was carried out using FITC Annexin V/Dead Cell Apoptosis Kit (Invitrogen™ Molecular Probes®, Eugene, OR, USA) as per manufactures protocol.

Network Construction

Collection of bioactive

The bioactive compounds of TC and ZO were collected from the UNPD (Universal Natural Product Database) in ‘.sdf’ file format and PubChem CIDs of the same were collected. This database allows free access to molecular structure and formula of bioactive (Chandran et al. 2015).

Prediction of targets

The ‘.sdf’ files having structures of bioactive were submitted to BindingDB database for predicting their target proteins. We retrieved targets by keeping score between 0.9 and 1. BindingDB is a public database of measured binding affinities and protein interactions considered to be drug-targets with ligands. The data from the binding database also gives information about experimental conditions such as kinetics, enzyme inhibition, EC50 values, etc. BindingDB currently contains about 1,200,000 binding data for 5500 proteins and over 520,000 drug-like molecules. Targets obtained from this database were linked to the UniProt database to obtain UniProt IDs and more information about the target proteins.

Target associated disease

Cancer-associated targets from the therapeutic target database (TTD) and the disease and gene annotation (DGA) were collected. By deleting the duplicates, uncompleted structures, structures with no active-site reported and the structures from the organisms other than Home sapiens, we retrieved 104 targets associated with diseases of Homo sapiens specifically from the databases.

Construction of the drug-target network

Tinospora cordifolia (TC) and ZO individual and combined networks were constructed using Cytoscape 3.2.1, Java-based open software. A network is made of nods, edges and the points of communication with two or more interconnecting entities such as a source of bioactive-target-disease. Different tools available in Cytoscape were used for the analysis of network and biological interactions. Here, we predicted the potential natural anticancer products, which could be good candidates for some malignancies based on the drug-target network.

Real-time PCR

Total RNA was isolated using TRIZOL reagent (Sigma). cDNA synthesis was carried out using a High Capacity cDNA conversion kit (Applied Biosystems). Gene expression analysis was done with SYBR green chemistry and ABI Step One Plus system (Applied Biosystems). Beta-actin(Shah et al. 2016), MMP9 (Qin et al. 2017) and ER-α (Rasoulzadeh et al. 2016) primers were used for real-time PCR as described previously. Results were analyzed using the delta–delta Ct method. All reactions were performed in triplicate from three independent experiments and data were expressed as mean ± SD.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 5.0 software. Experiments were performed in triplicates. Data were analyzed by One way or Two-way ANOVA, followed by “Bonferroni’s Multiple Comparison Test” to determine significance.

Results

Anti-proliferative effect of TC, ZO, and TC + ZO

Tinospora cordifolia (TC), ZO, and TC + ZO were tested in MCF-7 human cancer cell line. Both the extracts individually and in combination showed anti-proliferative activity at different concentrations ranging from 1 ng ml−1 to 100 μg ml−1. TC and ZO both showed 67% viability at 100 μg ml−1 on MCF-7 cell line as shown in (Fig. 1a and b). TC + ZO combination showed 55% viability on MCF-7 cells at 1 μg ml−1 as shown in (Fig. 1c). Neither TC nor ZO showed much reduction in percent viability of MCF-7 breast cancer cells at concentration 1 μg ml−1. TC showed an IC50 value of 509 μg ml−1 and ZO showed an IC50 value of 1 mg ml−1. However, when they are used in combination showed the IC50 value of 2 μg ml−1. Tamoxifen, a standard drug, showed a reduction in cell growth with an increase in concentration (Fig. 1d).

Fig. 1.

Fig. 1

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Effect of aqueous extract of different concentrations of TC (a), ZO (b), combination (c) and tamoxifen (d) on the proliferation of MCF-7 cell line was assessed using MTT assay. Data are expressed as Mean ± SEM. Data are analyzed by “one-way analysis of variance” followed by “Bonferroni’s Multiple Comparison Test” (*p < 0.05, **p < 0.01)

Tinospora cordifolia + Zingiber officinale (TC + ZO) inhibits BrdU incorporation

BrdU incorporation assay was used to study the inhibition of DNA synthesis to confirm anti-proliferation activity in MCF-7 cells treated with TC, ZO, TC + ZO, and standard drug tamoxifen. TC, ZO and TC + ZO showed 73 ± 1.2%, 84 ± 1.2% and 68 ± 2.8% BrdU incorporation at 1 µg ml−1, respectively. Tamoxifen showed 85 ± 1.3% BrdU incorporation at a concentration of 5 µg mL−1. TC + ZO combination showed a significant decrease in incorporation of BrdU at 1 μg ml−1 concentration as compared to tamoxifen (Fig. 2). BrdU incorporation was lowest in TC + ZO combination at 1 μg ml−1 concentration, suggesting that the antiproliferative effect is due to the reduction of DNA synthesis.

Fig. 2.

Fig. 2

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Effect of TC, ZO, and TC + ZO on BrdU incorporation during DNA synthesis of the MCF-7 cell line. TC + ZO inhibit DNA synthesis significantly at the concentration of 1 μg ml−1 and 10 μg ml−1. Data are represented by Mean ± SEM. Data are analysed by “two-way analysis of variance” followed by “Bonferroni’s Multiple Comparison Test” (***p < 0.001; **p < 0.01; *p < 0.05)

Tinospora cordifolia + Zingiber officinale (TC + ZO) arrest cell cycle at G0/G1 phase

Cell cycle analysis was performed using propidium iodide and percentage of MCF-7 cells in G0/G1, S, and G2/M phase was determined and the cell cycle histogram was represented in (Fig. 3a–e). Treatment with TC and TC + ZO showed 65 and 71% accumulation of cells in G0/G1 phase, respectively (Fig. 3f). ZO and reference compound Tamoxifen showed 30% cells in G2/M phase. TC + ZO combination showed significant cell cycle arrest in G0/G1, indicating the possible alteration of cyclin A and B. All the treatments were found to be static for distribution of DNA content in the S phase.

Fig. 3.

Fig. 3

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Effect of TC, ZO and TC + ZO combination on cell cycle regulation of MCF-7 cells. Distribution of percentage of cells in G0/G1, S and G2/M phases when treated with medium (a), ZO (b), TC (c), TC + ZO (d), and TAM (e) was represented in the histogram. Combined representation of cell cycle phases of MCF-7 cells was assessed by FACS analysis (e). Data are analyzed by “two-way analysis of variance” followed by “Bonferroni’s Multiple Comparison Test” (***p < 0.001; *p < 0.05)

Tinospora cordifolia + Zingiber officinale (TC + ZO) induces anti-migration potential

The effect of TC, ZO, TC + ZO, and tamoxifen on wound healing is shown in (Fig. 4a–e). After 24 h treatment, percentage area covered by cells was lowest for TC + ZO, i.e. 4.7%, followed by tamoxifen 11.3% and ZO 13.2% (Fig. 4f), indicating anti-migration potential of TC + ZO combination.

Fig. 4.

Fig. 4

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Effect of medium (a), ZO (b), TC (c), TC + ZO (d), and tamoxifen (e) on MCF-7 cell migration. Percent area healed by cells after treatment for 24 h measured by wound scratch assay (f). Distance between scratched areas was measured by Zeiss Inverted microscope using Motic Image plus 2.0. TC + ZO showed significantly less percent area (4.7%) covered by the cell as compared to ZO (13%), TC (22%) and tamoxifen (11%). Data are analyzed by “one-way analysis of variance” followed by “Bonferroni’s Multiple Comparison Test” (***p < 0.001; *p < 0.05)

Tinospora cordifolia + Zingiber officinale (TC + ZO) combination treatment induces apoptosis and necrosis in MCF-7 cells

Annexin V is a phosphatidylserine-binding protein which interacts strongly with phosphatidylserine residues on the outer membrane surface leading to green fluorescence. Treatment with TC showed apoptotic cells at 1 μg ml−1 (Fig. 5b) and with ZO showed apoptotic and necrotic cells at 1 μg ml−1 (Fig. 5c) as compared to the control (Fig. 5a). Highest necrotic cell death was observed when cells are treated with TC + ZO combination1 μg ml−1 (Fig. 5d). Tamoxifen also induced apoptosis in MCF-7 cells at 5 µM concentration (Fig. 5e) wherein propidium staining was less observed after treatment.

Fig. 5.

Fig. 5

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Immunofluorescence analysis of MCF-7 cells by annexin V and propidium iodide expression after exposure to medium (a), TC-1 μg ml−1 (b), ZO-1 μg ml−1 (c), TC + ZO-1 μg ml−1 (d), and tamoxifen-5 µM (e) for 24 h. The red arrow represents propidium iodide stained dead cells, green arrow represents-apoptotic cells

Network pharmacology approach to understanding the pharmacological and anticancer mechanism

Plant-disease network

The network of TC and ZO with a response to various diseases was constructed as shown in (Fig. 6). We found that TC showed a critical role in most common diseases such as cardiac disease, psychiatric disease, metabolic disorder, and arthritis (Fig. 6a). ZO showed potent role in diseases like cardiac, inflammatory and metabolic disorders (Fig. 6b). TC and ZO showed fewer interactions with arthritis, Alzheimer’s disease, neurological disorders, sepsis, etc. Interestingly, both the plants showed maximum interactions with cancer, which is in line with the above in vitro results. Hence, we have carried out the construction of sub-networks in detail to explore the mechanism.

Fig. 6.

Fig. 6

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TC (a) and ZO (b) individual network of all diseases: TC shows maximum edges connecting to cancer followed by psychiatric disease, cardiac disease, and metabolic disorders. In ZO, cancer showed maximum edges followed by cardiac disease, metabolic disorder, and inflammatory disease. The pink circle at the canter shows the source plant and they are connected to diseases by edges representing direct physical interaction

Prominent targets modulated by bioactives from TC and ZO

Prominent targets modulated by bioactives from TC were CYP17A1, AR, NR1H3, HMGCR, ACHE, MMP2, MMP9, CA2, OPRK1, F3, ESR1, and ESR2,whereas those from ZO were CYP17A1, AR, NR1H3, HMGCR, ESR1, ESR2, PRKCA, CA2, ALOX5, APP, and STK33. The detailed UNPD IDs of bioactives and their sources are represented in Supplementary file (Fig. S1) and respective targets are listed in the Supplementary file (Table S1).

Tinospora cordifolia (TC)-bioactive-targets in cancer

MMP2 and MMP9 showed maximum interactions as compared to HMGCR, ESR2, NR1H3, CYP17A1, ACHE, OPRK1, F3, and CA2. MMP2 is associated with 11 and MMP9 is associated with 9 types of cancers. Both genes are involved commonly in the prostate, hormone-refractory prostate, lung, pancreatic, Kaposi’s sarcoma and renal cell carcinoma (Fig. 7). Central pink V shape indicates the source and the surrounding rectangles indicate bioactives present in TC. Further, blue diamonds indicate the target genes and red triangles indicate the associated specific type of cancer. MMP2 specifically showed single interaction with breast cancer, skin cancer, hepatocellular and cancer unspecific, whereas MMP9 interacted with advanced lung cancer and brain cancer. N-trans-Feruloyltyramine from TC has an affinity towards MMP2 and MMP9. But we did not observe any bioactives from ZO interacting with MMP2 and MMP9 based on our selected score of 0.9–1 when used in combination and alone.

Fig. 7.

Fig. 7

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A pharmacological network of Tinospora cordifolia (A): F3, OPRK1, CA2 are connected with fewer nodes via edges as compared to ACHE, MMP2, MMP9 NR1H3, etc. Frequency of nodes is high between renal cell carcinoma, non-small cell lung cancer, lung cancer, prostate cancer, etc. but found to be less in glioma, focal ischemia, cervical cancer, etc. Note: Pink V shape: source; green round rectangles: bioactives; blue diamond: target; red triangle: type of cancer

Zingiber officinale (ZO)-bioactive-targets in cancer

ALOX5 shows maximum interactions as compared to PRKCA, APP, CA2, STK33, ESR1, NR1H3, ESR2, HMGCR, CYP17A1, and AR. It shows six interactions with pancreatic, urological, gastrointestinal, cervical, chronic myeloid leukemia and bronchiolar carcinoma. Also, ALOX5 interacts with seven bioactives from ZO as shown in (Fig. 8). STK33 shows single interaction with bladder cancer, whereas APP and CA2 show dual interaction with pancreatic cancer. ALOX5, STK33, APP, and CA2 have seven common interactions with the bioactives 1-dehydro-[6]-gingerdione, 1-dehydro-[10]-gingerdione, 1-dehydro-[8]-gingerdione,[10]-dehydrogingerdione, [8]-dehydrogingerdione, [6]-dehydrogingerdione and curcumin.

Fig. 8.

Fig. 8

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Pharmacological network of Zingiber officinale (B): Shows maximum frequency of interaction with ALOX5, APP, and STK33 followed by ESR1, ESR2, NR1H3, CYP17A1, etc. In comparison with other cancers pancreatic, cervical, breast showed higher connections with nodes via edges. Note: Pink V shape: Source; green round rectangles: bioactives; blue diamond: target; red triangle: type of cancer

Tinospora cordifolia + Zingiber officinale (TC + ZO) bioactive-target network

In a merged network, a total of 16 genes were found to be regulated when used in combination of 18 bioactive compounds. Genes APP, MMP9, MMP2, CA2, ACHE, ALOX5, and STK33 showed the maximum number of interactions with bioactives, as well as cancers (Fig. 9). We have shortlisted three candidate genes, ALOX5, MMP2, and MMP9, based on the maximum interactions in the combined network. ALOX5 is involved in the regulation of urological cancer, bronchiolar carcinoma, pancreatic cancer, cervical cancer, chronic myeloid leukemia, and gastrointestinal cancers. MMP2 and MMP9 showed common interactions with 9 types of cancers (Fig. 9). As per our network, 10 bioactives from TC interacted with 10 genes having a vital role in 21 different types of cancers. Combination of TC and ZO results in the regulation of a set of targets associated with respective cancer types as listed in (Table S1).

Fig. 9.

Fig. 9

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Combined pharmacological network of Zingiber officinale and Tinospora cordifolia (C): MMP2, MMP9, ALOX5, ACHE, APP, CA2, and STK33 show a higher number of connected edges than OPRK1, PRKCA, CYP17A1, and HMGCR. Prostate, pancreatic and breast cancer show maximum interactions. Note: Pink V shape: source; green round rectangles: bioactives; blue diamond: target; red triangle: type of cancer

From ZO, 9 bioactives interacted with 11 genes showing association with 15 types of cancers. Interestingly, when both the plants were used in combination, a total of 18 bioactives regulated 16 genes involved in 27 types of cancers.

Effect of TC + ZO treatment on mRNA levels of MMP9 and ER-α gene

The MMP9 found to be downregulated by 1.2-fold and 1.4-fold by TC and ZO treatment respectively. Tamoxifen also found to downregulate the MMP9 expression by 1.2-fold whereas none of the above treatment expects TC + ZO combination altered the significant change in ER-α expression (Fig. 10). MMP9 gene showed significant downregulation by fivefold after TC + ZO treatment of MCF-cells, whereas the ER-α gene showed significant upregulation by 3.5-fold (Fig. 7). The fold change values were normalized using beta-actin as a housekeeping gene. MMP9 and ER-α showed a highly significant change in mRNA expression when treated with TC + ZO.

Fig. 10.

Fig. 10

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Effect of TC, ZO, TC + ZO and tamoxifen treatment on mRNA expression levels of MMP9 and ER-alpha on MCF-7 cells at 1 µg ml−1. Comparison of values between the control group and treated group was analyzed by “two-way analysis of variance” followed by “Bonferroni’s Multiple Comparison Test” (***p < 0.001; *p < 0.05)

Discussion

There are many natural phytochemicals have been reported previously for anti-cancer activity. Some of them have been well studied and progressing towards the market as an anticancer agent. But, the majority of them have been failed due to lack of bioavailability and unavailability of physicochemical properties (Gupta and Sharma 2018). Hence the detailed investigation using high-throughput techniques is needed. Interestingly, curcumin an ingredient of ZO causes downregulation of COX-2, iNOS, 5-LOX, EGFR (Gupta et al. 2017) and MMP2 (Gupta et al. 2017) expression and upregulation of p21 expression (Yarla et al. 2017) via multiple signalling pathways (Yarla et al. 2017) in several cancers (Gupta et al. 2017). Another ingredient of ZO is a gingerol which reduces the iNOS and TNF-α expression via nuclear translocation of NF-κβ. It also induces mitochondrial-mediated apoptosis cell death (Gupta et al. 2017) in ovarian, colon, leukemia, pancreatic and breast cancer (Gupta et al. 2017). The beauty of natural products such as curcumin is that it would not cause a cytotoxic effect on healthy cells (Gupta et al. 2017).

In the present study, we have used in silico network pharmacology: one of the systems biology approach to evaluate the biological activity of selected herbs. Combination of TC + ZO showed significantly higher inhibition of cell proliferation as compared to individual TC or ZO in a dose-dependent manner. Synergistic effect of TC + ZO is possible because of bioactives such as berberine, palmitine, beta-sitosterol, curcumin, etc. The anti-proliferative effects were further confirmed using the integrity of DNA by BrdU incorporation.

TC + ZO inhibited the cell cycle at the G0/G1 transition phase. Previously, ZO is reported to reduce the proliferation of skin carcinoma and colon cancer cells via G0/G1phase arrest, probably through inactivation of cyclin D1 (Quan et al. 2016). Tamoxifen arrests the cell cycle in G2/M phase. The key cyclins, cyclin A and cyclin B, bind to cdc2 in G2/M transition phase and remain functional (Vermeulen et al. 2003). The alteration of these key regulators leads to cell cycle arrest in the G2/M phase, preventing them from entering into S phase.

We have observed significant anti-migration potential of TC + ZO in MCF-7 cell line based on the percent area covered by the cells. MMP9 (Shay et al. 2015)and VEGF has a significant role in the proliferation, invasion, and migration of cancer cell line (Li et al. 2017c). Although we could not identify VEGF as a target, MMP2, MMP9, and ALOX5 genes were found to be involved in breast cancer progression through network analysis.

A herbal combination of ZO and Curcuma longa was found to be more effective than either in prostate cancer cells (Kurapati et al. 2012). Another combination of ZO, Curcuma longa and Allium sativum induced apoptosis in breast cancer cells (Vemuri et al. 2017). We are the first to report the synergistic effect of TC and ZO in cancer. Network analysis provided a new approach to rediscover the synergistic effect of the traditional formulation of TC + ZO in cancer.

The integration and utilization of novel analytical techniques open an opportunity to understand the multiple molecular effects of ayurvedic medicines at metabolite, gene and protein levels (Fauzi et al. 2013). Various cell signaling pathways such as MAPK, phosphorylation process of anti-apoptosis (Yang et al. 2014; Zhang et al. 2014), EGFR, Estrogen, Wnt signaling pathway (Li et al. 2017b) and synergism (Sun et al. 2015) were explored using the NP approach. We also used a similar approach to study the mechanism behind the synergistic antiproliferative effect of TC + ZO combination.

Mechanisms of combinations of the two herbs were analyzed in the treatment of several cancers based on NP. Both the plants, when used in combination, regulate a total of 16 genes and 27 types of malignancies.

Experimental validation of network analysis was carried out by MMP9 and ER-α gene expression analysis on MCF-7 cells treated with TC, ZO, and TC + ZO. Significant downregulation of MMP9 gene expression by fivefolds and upregulation of the ER-α gene by 3.5-fold demonstrates that the synergistic anti-proliferative, anti-migration activity of TC + ZO is due to modulation of MMP9 and ER-α genes. Numerous studies reported the over expression of MMP9 associated with poor prognosis in a variety of cancers (Köhrmann et al. 2009; Yousef et al. 2014; Majumder et al. 2019). Downregulation of MMP9 by boswellic acid and plumbagin were reported previously by Yarla et al. (2017), but they have not revealed the effect on an intermediate target. TC + ZO combination reduces MMP9 mRNA level significantly with modulation of its downstream target ER-α. Loss of ER-α strongly augments breast cancer invasiveness in MCF-7 cells (Afratis et al. 2017), whereas its expression with disease prognosis has not been fully characterized. Modulation of ER-α is necessary to reduce the proliferation of breast cancer cells (Xu et al. 2017). Loss of ER-α function and mutation induces metastasis (Bouris et al. 2015), the whereas-α expression is retained in lung, bone, lymph node metastases (Saha Roy and Vadlamudi 2012). ER-α is associated with tamoxifen resistance in ER-α positive tumors (Diao et al. 2016). The anti-migration activity of TC + ZO could be the result of an increase in ER-α expression.

In conclusion, network analysis provided a new approach to rediscover the synergistic effect of the traditional formulation of TC + ZO in several cancers. Further, cell-based in vitro assays and gene expression study supported the synergistic activity of Tinospora cordifolia and Zingiber officinale in hormone receptor-positive breast cancer. In vivo study on cancer models using the TC + ZO combination treatment will be carried out in our laboratory.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 443kb) (442.2KB, pdf)

Acknowledgements

Authors strongly acknowledge Prof. Bhushan Patwardhan (Vice Chairman, UGC, New Delhi, India) for enormous guidance and support throughout the study. We are also grateful to Dr. Uma Chandran for guidance during network construction and analysis. Further, we thank Dr. Niraj, Dr. Vaibhav, Dr. Tejas, Mr. Avinash, Ms. Kavita and Ms. Jyoti for their assistance.

Abbreviations

MMP2

Matrix metalloproteinase 2

MMP9

Matrix metalloproteinase 9

ALOX5

Arachidonate 5-lipoxygenase

ESR1

Estrogen receptor 1

ESR2

Estrogen receptor 2

ER-α

Estrogen receptor alpha

HMGCR

3-hydroxy-3-methylglutaryl-coenzyme A reductase

ACHE

Acetylcholinesterase

PRKCA

Protein kinase C alpha type

CA2

Carbonic anhydrase 2

OPRK1

Kappa-type opioid receptor

AR

Androgen receptor

CYP17A1

Steroid 17-alpha-hydroxylase/17,20 lyase

NR1H3

Oxysterols receptor LXR-alpha

F3

Tissue factor

STK33

Serine/threonine-protein kinase 33

APP

Amyloid beta A4 protein

Author contributions

KJ designed the research plan, GJ performed all the experiments. KJ and GJ wrote the MS.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Compliance with ethical standards

Conflict of interest

Authors declare that there is no conflict of interests.

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