Potentiation of paclitaxel activity by curcumin in human breast cancer cell by modulating apoptosis and inhibiting EGFR signaling
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- Zhan, Y., Chen, Y., Liu, R. et al. Arch. Pharm. Res. (2014) 37: 1086. doi:10.1007/s12272-013-0311-3
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It has been suggested that combined effect of natural products may improve the treatment effectiveness in combating proliferation of cancer cells. Here, we examined the combined anticancer activities of compounds of three natural origin including baicalein, curcumin, and resveratrol with chemotherapy drug paclitaxel respectively, which showed that combination of paclitaxel with curcumin exhibited synergistic growth inhibition and induced significant apoptosis in MCF-7 cell lines. Treatment of MCF-7 cell lines with paclitaxel and curcumin induced the apoptosis of regulatory protein Bcl-2 but decreased Bax expression. In addition, simultaneous treatment with paclitaxel and curcumin strongly inhibited paclitaxel-induced activities of EGFR signaling. Furthermore, the combination of paclitaxel and curcumin exerted increased anti-tumor efficacy on mouse models. Overall, our data described the promising therapeutic potential and underlying mechanisms of combining paclitaxel with curcumin in treating breast cancer.
KeywordsBreast cancerPaclitaxelNatural productsSynergismApoptosisEGFR signaling
Breast cancer is a leading cancer in women and despite the benefits of the current therapies a significant number of patients with this tumor are at risk of relapse. Currently, chemotherapy anti-cancer drugs have been proven to be effective in clinical settings. Paclitaxel is the drug of choice as it exhibits significant antitumor activity toward breast cancer among others (Perez 1998; Sparano et al. 2008). However, the success of paclitaxel chemotherapy in cancer patients is limited by myelotoxicity and neurotoxicity (Maier-Lenz et al. 1997). It is necessary to find new chemotherapeutic strategies in breast cancer cells that provide higher tumor response and lower toxicity. The combined treatments with several chemotherapy regimens or even chemopreventive medicine are often used not only for the enhancement of the treatment effect, but also to reduce the toxicity of these medicines.
Natural products have played a significant role over the years in the development of anticancer drugs as more than 60 % of the drugs are of natural origin (Gupta et al. 2010; Newman et al. 2003). Baicalein, curcumin and resveratrol are such agent with multiple pharmacological activities. Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one) is a major flavonoid isolated from Scutellaria baicalensis, which is a herb that has been used extensively in traditional Asian medicine (Li-Weber 2009). Baicalein has an inhibitory effect on skin cancer (Wu et al. 2011), colorectal cancer (Huang et al. 2012), breast cancer (Wang et al. 2010), prostate cancer (Miocinovic et al. 2005), and bladder cancer etc. (Chao et al. 2007). Baicalein was recently reported to induce apoptosis by Mcl-1 down-regulation in human pancreatic cancer cells (Takahashi et al. 2011).
Curcumin, a natural yellow pigment present in the rhizome of Curcuma longa, is widely used as a spice and food-coloring agent. In recent years, this agent has been shown to have a significant inhibitory effect against the development of various cancers, including colon cancer, head and neck squamous, advanced pancreatic cancer, cervical cancer and breast cancer (Clark et al. 2010; Dhillon et al. 2008; Johnson and Mukhtar 2007; Nagaraju et al. 2012). Curcumin has been shown to suppress activation of NF-κB, STAT-3 and the dynamic instability of microtubules (Aggarwal 1995; Banerjee et al. 2010; Saydmohammed et al. 2010). It has also been shown to downregulate the expression of genes such as cyclooxygenase-2, matrix metalloproteinase-9, cyclin D1 and the adhesion molecules (Shehzad et al. 2010).
Resveratrol (3,4′,5-trihydroxy-trans-stilbene) is highly enriched in a variety of food sources, such as grapes, berries, peanuts, red wine, and other plant sources. A number of previous studies showed that, resveratrol were linked to myriad physiological benefits, including protection against cardiovascular disease, cancer, age-related deterioration, and the pathological consequences of high-fat diets (Frbmont 2000). Extensive data in human cell cultures indicate that resveratrol can modulate multiple pathways involved in cell growth, apoptosis, and inflammation (Athar et al. 2007; Chen et al. 2010).
These established results have led us to hypothesize that these active compounds may possibly improve the effectiveness of the traditional anti-cancer drugs.
Therefore, in this study, we investigated the combined effects of three natural origin compounds with paclitaxel on the breast cancer cell line MCF-7. The results of this study may also provide evidence for the treatment of breast cancer.
Materials and methods
Chemicals and regents
Baicalein, curcumin, resveratrol and paclitaxel were obtained as powder with a purity of >98 % from the National Institutes for Food and Drug Control. Cell culture mediums (RPMI 1640), trypsin, MTT, DMSO, RNase, PI, Annexin-V-FITC, Hoechst 33258 were purchased from Sigma (St. Louis, MO, USA). Reactive Oxygen Species Assay Kit was from Beyotime Institute of biotechnology. Akt, p-Akt, ERK1/2, p-ERK1/2, EGFR, p-EGFR antibody were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Bcl-2, Bax, and HRP-conjugated GAPDH monoclonal antibody were purchased from Proteintech Group (Chicago, IL, USA). Anti-rabbit antibody was purchased from Santa Cruz Biotechnology (CA, USA).
Cell culture and animals
Human breast cancer cell line MCF-7 was obtained from Shanghai Institute of Cell Biology in the Chinese Academy of Sciences and cultured in RPMI1640 supplemented with 10 % heat-inactivated fetal bovine serum (Lanzhou National Hyclone Bio-engineering Co., Ltd, China). Cells were maintained in an incubator with a humidified atmosphere containing 5 % CO2 at 37 °C. Murine sarcoma S180 cell line were obtained from Fourth Military Medical University (Xi’an, China) and preserved in our laboratory.
Six-week-old male mice were obtained from the Experimental Animal Center of the Xi’an Jiaotong University. Animals were maintained under specific pathogen-free conditions and had access to sterile food and water. All animal experiments were performed according to the guidelines and approval of the Institutional Animal Care and Use Committee of Xi’an Jiaotong University.
Measurement of IC50s for Baicalein, curcumin, resveratrol and paclitaxel
The effect of baicalein, curcumin, resveratrol and paclitaxel on MCF-7 cell proliferation was evaluated by the MTT assay. Exponentially growing cells were seeded into 96-well plates at a density of 2 × 104 cells per well in medium. After 24 h incubation at 37 °C, cells were treated with these agents at various concentrations for 48 h. Then, 20 μl of MTT (5 mg/ml) was added to each well and incubated at 37 °C for 4 h. After the removal of medium, 150 μl of DMSO was added to each well, and the optical density of cells was determined with a microplate reader (Bio-RAD Instruments, USA) at 490 nm and expressed as absorbance values.
Exponentially growing MCF-7 cells were harvested and plated (2 × 104 per well) in 96-well plates and incubated at 37 °C, 5 % CO2 for 24 h. A range of drug concentrations were then added either concomitantly or sequentially at their fixed ratio based on their respective individual IC50 values for 48 h. The fractional inhibition of cell proliferation was calculated by comparison to control. After incubation for 48 h, cell growth was determined using an MTT assay.
The effects of the combination were calculated for each experimental condition using the combination index (CI) method based on the median-effect analysis of Chou and Talalay (1984). The CI is defined by the following equation: CI = (D)1/(Dx)1 + (D)2/(Dx)2 + α(D)1(D)2/(Dx)1(Dx)2, in which (Dx)1 and (Dx)2 are the concentrations of the combination required to produce a fu, and (Dx)1 and (Dx)2 are the concentrations of the individual drugs required to produce fu. α = 0 when the drugs are mutually exclusive (i.e., with similar modes of action), while α = 1 when they are mutually non-exclusive (i.e., with independent modes of action). CI > 1, CI = 1, and CI < 1 indicates synergy, additivity, and antagonism, respectively.
The migration assay was performed on a polycarbonate membrane (8-μm pore size) in a culture insert (Millipore, USA). MCF-7 cells were plated into the upper chamber of the insert with or without drugs, after incubation for 48 h, the insert was placed into a well of a 12-well plate containing 2 ml medium (supplemented with 10 % FBS), after 24 h, the top surface of the insert was scraped using a cotton swab and the cells on the lower surface of the membrane were fixed for 15 min with methanol and stained with 0.2 % crystal violet. Cells that had migrated to the bottom of the membrane were counted under a light microscope (magnification 40×). For each replicate (n = 3), cells in six randomly selected fields were counted and averaged. Data are expressed as a ratio to the control group.
Hoechst staining assay
To visualize apoptotic cell death and nuclear morphology, cells were stained with Hoechst staining. The number of apoptotic cells was measured by assessing the percentage of cells displaying chromatin condensation compared to the total amount of cells. Briefly, MCF-7 cells were seeded in 6-well plates and then treated with paclitaxel and curcumin individual or in combination at various concentrations respectively. After treatment for 48 h, cells were collected, washed by PBS and allowed to dry on slides. Genomic DNA was stained with Hoechst 33258 for 10 min at 37 °C according to the manufacturer’s instructions.
Flow cytometer analysis
MCF-7 cells were treated with paclitaxel and curcumin individuals or in combination at various concentrations for 48 h respectively. The cells were then collected, washed, and resuspended in PBS. The apoptotic cell death rate was examined with Annexin V-FITC and PI double staining (5 μl Annexin V-FITC, 10 μl PI for 15 min away from light) according to the manufacturer’s instructions. After staining the cells with Annexin V-FITC/PI, cell suspension was analyzed by the flow cytometer.
Detection of intracellular ROS
MCF-7 cells were seeded in six-well plates and treated with serial dilutions of the paclitaxel and curcumin individual or in combination for 48 h, and then incubated with 20 μM 2′,7′-DCFH-DA for 30 min. After washing with PBS twice, fluorescence intensity was measured in a microplate reader with excitation at 485 nm and emission at 535 nm.
Adherent cells were grown to 30–50 % confluence and then exposed to different agents, at various concentrations as described. After exposure, cells were harvested, washed with ice-cold PBS, lysed in 150 μl cold RIPA lysis buffer containing protease inhibitor cocktail and phosphates inhibitor cocktail, left on ice for 30 min, and then centrifuged at 12,000×g for 10 min at 4 °C; the supernatant was stored at −70 °C for later use. Protein concentration for each lysate was determined using the BCA protein assay (Sigma-Aldrich). The protein lysates were resolved by SDS-PAGE, and separated proteins were transferred to PVDF membranes and blocked with 5 % skimmed milk for 2 h. Then, the membranes were incubated with specific primary antibodies overnight at 4 °C and incubated with the relevant secondary antibodies at 1:10,000 dilutions at room temperature for 2 h in accordance with the manufacturer’s instructions. Finally, the blots were detected by Immobilon® Western (Millipore Corporation, MA, USA).
In vivo studies
All studies were approved by the institutional animal care and use committee and were carried out in accordance with institutional guidelines for animal care. Female Kunming mice (4–6 weeks old) were injected with 4 × 106 S180 cells by intraperitoneal injection. After 5 days culture, ascites was extracted, resuspended in 5 % saline and transplanted into the right axilla of the mouse, next, animals were assigned to five treatment groups at random and administrated with paclitaxel and curcumin individual or in combination daily respectively while control animals received equivalent volumes of dissolvant. After a total of 10 days treatment, mice were sacrificed and tumor tissue was removed, weighed.
All data were obtained from at least three independent experiments and expressed as mean ± SEM. Comparisons of the different groups were performed with Student’s t test. P < 0.05 was considered the minimal level of significance.
IC50s for baicalein, curcumin, resveratrol and paclitaxel individual on MCF-7 cells
Growth inhibitory effects of paclitaxel in combination with baicalein, curcumin or resveratrol
To explore whether baicalein, curcumin or resveratrol could enhance the effects of paclitaxel currently used to treat breast cancer, MCF-7 cells were simultaneously and continuously exposed to increasing equitoxic concentrations of paclitaxel and these three agents singly or in combination, and cell viability was measured. When the combination effects were studied, the cells were exposed for 48 h to both of the two drugs concurrently at a fixed ratio approximating their individual IC50s, so that the contribution of anti-proliferative effect for each compound in the combinations is roughly the same.
Summary of CI values at 20, 50 and 80 % fraction affected
CI at fraction affected (%)
BAI + PTX
CUR + PTX
RES + PTX
Because paclitaxel and curcumin interacted synergistically to inhibit breast cancer cell growth, whereas the combination of paclitaxel and baicalein or resveratrol, just showed antagonism. We further investigated the effect and mechanism of paclitaxel and curcumin in combination on breast cancer MCF-7 cells.
Paclitaxel in combination with curcumin inhibited tumor cell migration
Paclitaxel in combination with curcumin induced tumor cell apoptosis
Paclitaxel in combination with curcumin generated ROS in MCF-7 breast cancer cells
Involvement of Bcl-2/Bax in the apoptotic process induced by paclitaxel in combination with curcumin
Curcumin inhibited EGFR activity induced by paclitaxel in breast cancer cells
In vivo study
In vivo inhibitory effect of curcumin in combination with taxol
CUR + PTX
CUR + PTX
CUR + PTX
25 + 5
75 + 5
225 + 5
Final tumor weight (g)
2.53 ± 1.09
1.89 ± 1.00
1.49 ± 0.62
1.38 ± 0.81
0.50 ± 0.48
Tumor growth inhibition (%)
Final body weight (g)
25.81 ± 4.35
24.05 ± 2.40
25.30 ± 2.37
24.25 ± 2.88
22.92 ± 2.27
Plants have been utilized as medicines for thousands of years. These medicines initially took the form of crude drugs such as powders, tea and other formulations. The use of medicinal plant was then developed into anticancer drugs. This involves the isolation and characterization of pharmacological active compounds. More recently, chemotherapeutic strategies have been moved on to the use of combined active compounds where they are believed to be more active as compared to the single agent itself. Therefore, the efficacy of treatment would increases and the possibility of toxic effect may be lowered due to the extremely low usage of drug. Baicalein, curcumin and resveratrol were the compounds isolated from plants and have been reported to have a wide spectrum of pharmacological activities. They also exhibited significant antitumor activities. Moreover, curcumin is currently involved in early phase of clinical trial as potential chemopreventive agent (Dhillon et al. 2008; Sharma et al. 2004). Hence, it is rational to investigate whether these compounds as the new antiproliferative agents can sensitize tumors to chemotherapeutic agent paclitaxel for breast cancer cells.
In this study, we investigated whether three plant-originated active compounds baicalein, curcumin, and resveratrol could enhance the antitumor actions of paclitaxel in human breast cancer cell lines MCF-7 in culture. Baicalein, curcumin, resveratrol and paclitaxel exhibited significant inhibitory effects on MCF-7 cells. A synergy interaction of the combinations of curcumin and paclitaxel was observed in MCF-7 cells, however, an antagonism interaction was observed in MCF-7 when paclitaxel was combined with baicalein and resveratrol respectively. Based on the above results, the mechanism of synergy interaction of the combinations of curcumin and paclitaxel was further determined in this study.
Study on the mode of cell death induced by the simultaneous treatment of combined paclitaxel and curcumin was conducted in this study. Apoptosis is a programmed cell death which is activated to expel damaged cells, excessive numbers of cells and cells that are not needed during the development and normal tissue homeostasis. Failing of trigger apoptotic cell death may lead to the development of neoplasia. Therefore, the cytotoxicity effect via the induction of apoptosis was considered as criteria for the identification or screening for a new cancer chemotherapy agent (Cotter 2009), and most chemotherapeutic drugs induce apoptosis in cancer cells.
To determine whether the synergistic antiproliferative effects of the drugs observed might be caused by a synergistic effect on apoptosis, the combination treatment was tested to induce apoptosis cell death. The Hoechst 33258 dye was able to diffuse through intact membranes of MCF-7 cells and stain their DNA. Using Hoechst staining we could distinguish apoptosis which was more intense in paclitaxel–curcumin combined treatment groups than the other groups. By using flow cytometer, we demonstrated that both paclitaxel and curcumin triggered apoptosis, whereas there was a marked increase of apoptotic index after treatment with paclitaxel–curcumin combination The above results indicated that curcumin enhanced the paclitaxel-induced apoptosis of breast cancer cells. To further clarify the mechanisms underlying combined paclitaxel–curcumin inducing apoptosis, we detected changes in bax and bcl-2, which were known to play a key role in the execution of apoptosis. In MCF-7 cell lines treated with curcumin and/or paclitaxel for 48 h, synergistic effect was observed for the two proteins, indicating that they were involved in the apoptosis induced by the combination of paclitaxel and curcumin. Thus, synergistic downregulation of bax and upregulation of bcl-2 might contribute to the synergistic effect in MCF-7 cell lines. This would at least in part explain the synergy effects of paclitaxel in combination of curcumin.
EGFR plays an important role in the growth and progression of breast cancer. EGFR-driven cell signaling contributes to the disease progression and cancer malignancy. Recent studies indicated that curcumin was able to inhibit the cancer cells growth by down-regulating EGFR protein expression and EGFR signaling (Sun et al. 2012). Our study found that paclitaxel activated EGFR in human breast cancer cells, treatment of breast cancer cells with curcumin completely abolished the paclitaxel induced EGFR activation. Curcumin also suppressed paclitaxel-induced ERK1/2 and AKT in breast cancer cells.
As in the in vitro studies, in the mouse transplanted tumor model we found that the curcumin and paclitaxel combination treatment significantly inhibited tumor growth more than the control or either one alone. It should be noted that the 5 mg/kg paclitaxel dose used was lower than doses previously shown to be effective against breast cancer in the mouse model (Nicoletti et al. 1993). Using this relatively less effective dose, we showed that the addition of curcumin potentiated growth inhibitory effect.
The experimental evidence from this study suggests that simultaneous treatment of paclitaxel and curcumin exhibits the synergistic antiproliferative effect and induces apoptosis towards human breast cancer cells MCF-7. The combination treatment induced apoptotic cell death as the experimental evidence from this study revealed that DNA condensation, increased intracellular ROS and DNA fragmentation were observed after the treatment. This synergism is associated with decreased expression of Bcl-2 and increased expression of Bax, as well as EGFR signaling blockade. Taken together, these preclinical data may provide an effective and a novel approach for paclitaxel chemotherapy, with less toxicity, in breast cancer patients.
This work was supported by National Natural Science Foundation of China (Grant Nos. 81302800 and 81102414), the National Science Foundation for Post-doctoral Scientists of China (Grant No. 2013M532062).
Conflict of interest