Ginseng (Panax quinquefolius) Augments Doxorubicin-Induced Inhibition of Human Breast Cancer Cell Proliferation and Tumor Growth: Mechanisms of Action

By: Laura L. Murphy, Mandy L. King, Kimberly A. Smith, LSI Edition 23

Laura L. Murphy, Mandy L. King, and Kimberly A. Smith 
Southern Illinois University School of Medicine, Department of Physiology, Carbondale, IL, USA

Introduction

Ginseng root extract inhibits the proliferation of human cancer cells in vitro, including those that represent many common cancers, such as breast cancer. Ginseng extracts contain over 20 different ginsenosides, saponin glycosides that are biologically active constituents of ginseng. Several ginsenosides have been identified that exert anticancer activity, including ginsenosides Rb1, Rc, Rg3, Rh2, and protopanaxadiol.

Because ginseng has been reported to have anticancer properties, as well as reputed to be a tonic that promotes general well-being, ginseng supplements may be taken by women with breast cancer who are undergoing chemotherapy. Doxorubicin (Adriamycin®) is a chemotherapy drug often used in the treatment of breast and other cancers. Dose-dependent doxorubicin actions on cancer cells include DNA disruption and apoptosis. The development of resistance to doxorubicin therapy is a common occurrence. Ginseng and ginsenosides have been reported to produce cancer cell linespecific effects that include inhibition of cell proliferation, altered MAP kinase cell signaling, and/or induction of apoptosis.

This study was designed to examine potential interactions between ginseng and doxorubicin, usingMCF-7 andMDA-MB-231 breast cancer cells that are either sensitive or resistant to doxorubicin, and to address the mechanisms of ginseng-doxorubicin action.

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Methods

Hot Water-extracted Ginseng

Powdered American ginseng root (courtesy of the Ginseng Board of Wisconsin) was mixed in distilled water (1:9) and subjected to vigorous shaking in a 90 °C water bath for one hr. The process was repeated, supernatant frozen and freeze-dried.

Cell Proliferation Assays

Wild-type or doxorubicin-resistant MCF-7 and MDA-MB-231 cells (resistance was generated by continuous exposure to increasing concentrations of doxorubicin) were either not treated (control) or were treated every 48 hr with ginseng extract and/or doxorubicin and were counted on Day 6 of treatment. Data are expressed as a percent of the average number of cells relative to control. IC50 concentrations were determined using linear regression from the plots of percentage growth inhibition versus the logarithm of the drug concentration.

Treating Mice and Monitoring Tumor Growth

Female HSD nu/nu athymic mice (Harlan Sprague-Dawley, Indianapolis, IN) at 6-8 weeks of age were used for the xenograft studies. Cells were inoculated s.c. into both flanks of each mouse and an estradiol pellet (0.72 mg/pellet, 60 day release, sc) was implanted. Approximately 2 weeks post-inoculation, animals were randomly assigned to treatment groups that included water only controls; 1% ginseng extract in lieu of drinking water, doxorubicin (Dox; 1.75 mg/kg, ip, 5×/week every 2 weeks); or ginseng + Dox. Animals not receiving Dox were injected with sterile saline. The ginseng extract was provided ad libitum for the duration of the study. The effects of treatments on tumor size were assessed using two-way ANOVA and Fisher’s post-hoc test. Differences were significant at p<0.05.

Antibody Microarray

Subconfluent cells were either not treated (control) or were treated daily for 24, 48 or 72 hr with ginseng extract (0.5 mg/mL), doxorubicin (5 ng/mL), or ginseng+doxorubicin. Differences in protein expression were detected using the Panorama® Antibody Microarray-Cell Signaling Kit. Protein was extracted from control (Con) or treated cells (Rx), labeled with Cy3/Cy5 dye, and the array assayed according to manufacturer instructions and with kit buffers. Briefly, 1mg/mL protein samples were added to a single monoreactive Cy3 or Cy5 dye pack (Amersham Life Sciences, Piscataway, NJ) and incubated for 30 min with mixing every 10 min. Free unconjugated dye was removed by centrifugation using the provided spin columns. The final labeled proteins were assayed again for protein concentration and dye incorporation measuring absorbance of diluted (1:100) samples at wavelengths of 552 nm (Cy3) and 650 nm (Cy5). If a dye to protein ration of ≥2 was obtained, dye swaps of the protein samples were made by combining 25 μg of Con-Cy3 with 25 μg of Rx-Cy5 (slide 1) and 25 μg of Con-Cy5 with 25 μg of Rx-Cy3 (slide 2). Each dye combination was added to 5 mL of provided incubation buffer and transferred to separate slides. After gently rocking at room temperature for 30 min, slides were then washed three times for 5 min each in provided wash buffer. Slides were rinsed with PBS and allowed to air dry. Slides were scanned using an Axon 4000B GenePix® array scanner and image analysis performed using GenePix Pro 5.0 Software. The average median of ratios was obtained for each antibody and the internally normalized ratios (INR) were calculated as the square root of the ratio of this value for slide 1 to slide 2. The mean INR was determined and significant changes in protein expression were considered to be those whose INR was greater (increased expression in Rx) or less (decreased expression in Rx) than two standard deviations from the mean INR.


Figure 1. Human breast cancer MCF-7 and MDA-MB-231 cells were treated for 6 days with a wide concentration range of water-extracted ginseng. Both estrogen receptor-positive MCF-7 and –negative MDA-MB-231 cells exhibited a concentration-dependent decrease in cell proliferation with IC50s of 0.34 and 0.67 mg/mL, respectively.


Figure 2. Cells were treated with doxorubicin (DOX; 1-50 ng/mL), alone or in combination with ginseng extract (GE; 0.5 mg/mL) for 6 days. In MCF-7 cells, the IC75s for DOX and DOX+GE were 21.2 and 4.9 ng/mL, respectively; in MDA cells, the IC75s were 7.7 and 2.5 ng/mL, respectively. The interactions between DOX and GE were synergistic for both cell lines as determined by combination index analysis.


Figure 3. Wild-type and doxorubicin-resistant MCF-7 and MDA-MB-231 cells were treated with doxorubicin (0.25-300 ng/mL), alone or in combination with ginseng extract (GE; 0.5 mg/mL) for 6 days. Note the ≥30-fold increase in resistance to doxorubicin in the drug-resistant cell lines. Co-treatment with GE reversed the resistant phenotype and rendered the cells sensitive to doxorubicin.


Figure 4. Female nude mice inoculated with either wild-type MCF-7 cells (left graph) or doxorubicin-resistant MCF-7 cells (right graph) were treated with ginseng extract (GE; 1% solution in lieu of drinking water), doxorubicin (Dox; 1.75 mg/kg, ip; 5×/week every 2 weeks), or Dox+GE. Control animals received untreated drinking water and ip saline injections. Data represent the average tumor size (N ≥ 12 animals per treatment group) at intervals postinitiation of Dox treatment. * = p<0.05 when compared to control group.


Figure 5. MCF-7 cells were treated with ginseng and doxorubicin, alone and in combination. Each column represents treatment of cells with ginseng, doxorubicin, or doxorubicin and gingseng combined. Blue arrows denote change in protein expression at 24 hr. Green arrows indicate change in protein expression at 48 hr. Black arrows show change in protein expression at 72 hr.

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Conclusions

Ginseng inhibits MCF-7 and MDA-MB-231 cell proliferation by altering cell signaling pathways that regulate the mitotic cell cycle as well as apoptosis. Ginseng increases doxorubicin-induced therapeutic effects and reverses doxorubicin resistance in breast cancer cells both in vitro and in vivo. The combination of ginseng and doxorubicin induces a number of changes in protein expression reflecting decreased G2/M arrest and increased cell death.

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