The anti-tumor effect of Erzhi pills on breast cancer cell MCF-7 and the intervention in endocrine therapy adverse reactions

Qi Liu, Long-Long Li, Chun-Hong Xu, Guang-Qiang Zhang, Zheng Sun, Ming-Ming Shi,Shi-Min Ji, Guo-Lou Li,*

1 Depatment of Breast and Thyroid Surgry, The Traditional Chinese Medicine Hospital of Weifang, Shandong, China；

2 Clinical College of Weifang Medical University, Shandong, China；

3 Depatment of General Surgry, The Traditional Chinese Medicine Hospital of Shouguang,Shandong, China.

Abstract Objective: The aim of this study was to investigate the potential ability of medicated serum of Erzhi pills on growth, proliferation and inflammation of the human breast cancer cell line MCF-7 cells.Methods: 3-month-old female Sprague–Dawley rats were used to prepared medicated serum, and randomly assigned into four groups: control group (distillated water), low doses (2.25g/kg/d),middle doses (4.5g/kg/d), high does (9.0g/kg/d). The growth curve was meas-ured by MTT method,the proliferation was examined by colony formation assay. Inflam-matory cytokines IL-1β, IL-18 and IL-6 was detected by Quantitative real time polymerase chain reaction (RT-qPCR). Western blot was used to examine the expression of NF-kB signal pathway.Results: The medicated serum of Erzhi pills could inhibit the growth and proliferation of breast cancer cell MCF-7, especially, in the group of middle doses and high does (P＜0.05). The protein expression of IKKα, IκBαand p-NF-κB were significantly down regulated. As well as the mRNA expression levels of Inflammatory cytokine ,such as IL-1β, IL-18 and IL-6,were decreased.Conclusion: Medicated serum of Erzhi pills played an anti-tumor role in breast cancer via inhibiting the growth and proliferation of MCF-7. Moreover, Erzhi pills could suppress in-flammatory responses from the NF κB signaling pathways, and reduced skeletal muscle pain caused by breast cancer endocrine therapy, and Erzhi pills may be a new target for the treatment of breast cancer.

Key words: Breast cancer, Erzhi pills, Growth, Proliferation, Inflammation

Background

Breast cancer, which had the highest incidence of malignant tumor in Chinese women, was increasing year by year, especially in younger women [1,2]. Breast cancer seriously affected women's health and even endangers their lives.At present, the treatment of breast cancer by domestic and foreign experts was mainly surgical treatment, combined chemotherapy,radiotherapy, endocrine therapy and targeted therapy. In recent years, the role of traditional Chinese medicine in the prevention and treatment of cancer had gradually emerged,which had attracted wide attention to clinicians.Breast cancer belonged to the categories of traditional Chinese medicine "stone carbuncle","breast rock", "breast nucleus", "breast ruff","jealous breast ", " breast poison", "pomegranate flower hair" and so on [3]. Its pathogenesis was characterized by the coexistence of internal deficiency and toxic accumulation. Internal deficiency was liver-kidney Yin deficiency,which was caused by the disharmony of the thoroughfare, depression of liver, anxiety of spleen, and stagnation of liver-qi. Toxic accumulation was phlegm turbid stagnant knot,stasis toxin deposition, gather agglomerate.Therefore, its treatment was mainly in Qinggan Yangyin, removed sputum and scattered knot.Erzhi pills was a prescription for regulating liver and kidney diseases, which was recorded in Standards of Diagnosis and Treatment by Wang Kentang, a doctor in Ming dynasty. Ingredients of Erzhi pills include fructus ligustri lucidi(wine) and ecliptase. It has the function of tonifying liver and kidney, nourishing Yin and hemostasis.

The adverse reaction to endocrine therapy, such as joint pain, arthritis, muscle pain and other symptoms, the etiological factor were medicine poison damage the liver and kidney and loss Yin-blood, the pathogenesis were liver and kidney Yin deficiency, kidney failing to nourish liver, yin deficiency with yang hyperactive, and muscles and bones dystrophy. Therefore, the Chinese medical dialectical therapy used more nourished liver and kidney, enrich water to moisten wood, nourish the heart to tranquilize and relieve muscles and bones pain, etc.

Endocrine treatment of breast cancer was a major part of estrogen receptor positive (ER)breast cancer patients. Tamoxifen, an estrogen receptor antagonist, was the first choice of premenopausal patients, while aromatase inhibitors (AIs), letrozole, anastrozole and ethimetam were preferred for post menopausal patients. Aromatase inhibitors, by inhibiting aromatise to block estrogen synthesis, had been applied to ER positive breast cancer patients with significant curative effect and could reduce about 50% recurrence rate. However,the application of AIs would bring a series of adverse reactions, especially, musculoskeletal pain which was the main adverse reaction leading to the interruption in treatment for patients, thus reducing the treatment compliance of patients. At present, the mechanism of the adverse reactions to endocrine treatment-related musculoskeletal pain was still unclear. Therefore,the purpose of this study was to investigate the effect of Erzhi pills on the biological behavior of breast cancer cells and the intervention in the adverse reaction to musculoskeletal pain caused by endocrine therapy.

Materials

Test drug

Erzhi pills, included Nvzhenzi (Ligustrum lucidum Ait) 500g and Mohanlian (Eclipta prosTCMLIBata) 500g, which was purchased from the pharmacy of traditional medicine,traditional Chinese medicine hospital of Weifang. Dosage and preparation methods of Erzhi pills in accordance with Pharmacopoeia of the People's Republic [4]. The detailed procedures: the medicinal materials were soaked with five times water for 30 min, decocted for 30 min, filtered with four layer gauzes, then four times water decocted with the same method,collected filtrate, two components combined to concentrate 1 g/ml, 4℃reserved for experiment.

Test animals

Twenty-four female SPF-grade Sprague-Dawley rats, aged 3 months, weight (250 + 20 g), were purchased from Beijing weitonglihua experimental animal center. Certificate number:SCXK (Beijing) 2012-0001.

Test reagent

Breast cancer cell line MCF-7 was purchased from Shanghai cell bank, Chinese academy of sciences. DMEM and fetal bovine serum were purchased from Solarbio, USA. The MTT kit was supplied by biyuntian biotechnology Co.,Ltd. The total RNA extraction kits (centrifugal column) were from tiangens biochemical technology (Beijing) Co., Ltd. Rabbit antihuman IKKα、IκBαand p-NF-κB monoclonal antibodies and rat anti-rabbit IgG antibodies and β-actin monoclonal antibodies were purchased from Abcam. Primers of IL-1β、IL-18、IL-6 and GAPDH were purchased from Shanghai gemma pharmaceutical technology Co., Ltd.cDNA reverse transcription adopts PrimeScript reverse transcription kit provided by Beijing kangwei century biotechnology Co., Ltd.Fluorescence quantitative PCR using SYBR Premix Ex TaqTM ℃ kit.

Methods

Preparation of Medicated serum and grouping Twenty-four Sprague-Dawley rats were randomly divided into four groups: control group (distilled water), low-dose group (2.25g/kg/d), middle-dose group (4.5g/kg/d) and highdose group (9.0g/kg/d), with 6 rats in each group. The rats were gavaged medicine twice a day for one week. Two hours after the last gavage, rats were anaesthetized with 10%chloral hydrate, and draw blood from abdominal aorta, then standing at room temperature for 2h, centrifugal for 10 min at 2000 r/min, take the serum, 56℃ water bath inactivated for 30 min, 0.22um microporous membrane filter out the bacteria, saved for spare at -80℃. When used, the control serum was diluted to the corresponding concentration.

Cell culture

Human breast cancer cell line MCF-7 was cultured at 37°C in an atmosphere containing 5% CO2, culture medium containing 10% fetal bovine serum and 10% green/streptomycin DMEM medium. All the experimental cells were in a logarithmic growth phase.

MTT colorimetry was used to determine the cell growth ability

The cells of different groups were seeded into 96-well plates (1000 cells/well) and six wells were repeated. The cells were incubated for 24 h and subsequently treated with 20 µl 5g/l MTT and incubated at 37°C for 4 h. The supernatants were washed and 150 µl dimethylsulfoxide was added to each well to terminate the reaction.The optical density (OD) values was detected at a wavelength of 570nm on an enzymelinked immune detector. The OD values was continuously measured for 5 days, and the growth curves were produced. The day was set as abscissa and the absorbance value as the longitudinal coordinate.

Ability of cell cloning was detected by plate cloning assay

The cells were collected as a single cell suspension and were seeded into 6-well plate, each group had three repeated wells and contained 100 cells/wells. The single cell suspension was incubated in a humidified incubator at 37°C under 5% CO2, until the cell colonies were visible by eye. The cells were washed twice with PBS and fixed in 100%methanol for 30 min at room temperature.Following fixation, the cells were stained with 0.5% crystal violet for 5 min, prior to washing and drying. Positive colonies, containing ＞50 cells, were observed under a microscope,images were captured and colony numbers were quantified.

Western blot was used to observed protein expression

Lysis Buffer was used to lyse cells and the total protein were extracted after shock centrifugation.BCA kit was applied to determine the total protein concentration. The equivalent amount of protein (30g) were added to buffer solution boiled and denatured. Then the protein was electrophoresis separated from 10% SDS -PAG and transferred to PVDF membrane, 5%skimmed milk powder blocked 1 h, join primary antibodies at 4℃ refrigerator, incubation overnight (IKKα 1∶1000, IκBα 1∶1000, p-NFκB 1:1000, β-actin 1∶1000). The membrane was washed by TBST for 30 min, and the secondary antibody was incubated for 2h at 1:1000, then detected by chemiluminescence.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

The total RNA of each group cell was extracted by the total RNA extraction kit of cultured cells/bacteria (centrifugal column).The extraction procedure was carried out according to the instructions. According to the Beijing kang biological technology Co., LTD reverse transcription kit instructions for reverse transcription, reaction conditions: 42 for 15 min incubation, incubated with the 85 for 5 min.

IL-1β:

5'-CAGACCGGACGCTACACC-3' forward

5'-GAACACGTCTTGTCCGACCA-3' reverse IL-18:

5'-AGCAGAAGTCAGCCAGGTA-3' forward

5'-TCGGCGCAAGTGTTATTGGGT-3' reverse IL-6:

5'-AGGCGGAACGTGGTGTGGCA -3' forward

5'-TGATCCGCCTTGGTCGTGTTCC-3'reverse

GAPDH∶

5'-GTCATCAATGGAAATCCCATCA-3'forward

5'-CCAGTGGACTCCACGACGTAC-3' reverse

Statistical analysis

All experiments test independently for three times. Statistical analyses were performed using SPSS 19.0 software (SPSS, Inc., Chicago, IL,USA) and all data are expressed as the mean ±standard error of the mean. A least significant difference t-test was used for two sample comparisons between two groups. P ＜ 0.05 was considered to indicate a statistically significant difference.

Results

The medicated serum of Erzhi pills inhibited the growth ability of breast cancer cell MCF-7

Effect of medicated serum on the growth of MCF-7 cells detected using an MTT assay.The results showed that compared with control group, different dose medicated serum of Erzhi pills could inhibit the growth ability of MCF-7 cells, especially in the middle and high dose group, the difference was statistically significant(P ＜ 0.05).(Fig. 1)

The medicated serum of Erzhi pills inhibited cellproliferation ability of breast cancer cell MCF-7

Clone formation assay was used to observe the proliferation ability, after 4 days cultivation, the cell clone number of control group, low dose group, middle dose group and high dose group were respectively 154.86 8.355, 109.43±11.688,40±5.354 and 25.14±3.848, with the increase of drug concentration, the number of cell clone gradually reduced. Especially, the middle and high dose groups were statistically different compared with control group (P ＜ 0.05). (Fig. 2)mRNA expression levels of related proinflammatory factors IL-1β,IL-18 and IL-6 were detected by RT-qPCR

RT-qPCR was performed to assess mRNA level of pro-inflammatory cytokines in breast cancer cells MCF-7. Levels of IL-1β, IL-18 and IL-6 were significantly reduced following treatment with middle and high groups (P ＜ 0.05). These results suggest that Erzhi pills may ameliorate breast cancer progression by suppressing the inflammatory response.( Fig. 3)

Figure 3. Inflammatory cytokines were determined via reverse transcription-quantitative polymerase chain reaction in MCF-7

Figure 4. Western blot assays were used to determine levels of IKKα, IκBα and p-NF- κB following treatment with different dose Erzhi pills serum

The medicated serum of Erzhi pills suppresses the inflammatory response from the IKK α /NFκ B signaling pathway.

In the present study, western blot was applied to detect the NF- κB signaling pathway.It was observed that levels of IKKα, IκBα and phosphorylated NF- κB were markedly decreased following the middle and high dose Erzhi pills serum treatment. ( Fig. 4)

Discussion

Breast cancer was the most prevalent malignant tumor in women over the world. The occurrence and development of breast tumors through complex and multisteps [5]. This process mainly included initiation, progression and metastasis,as well as a series of self-renewal apoptosis,cell cycle and mobile events [6]. Breast cancer molecular subtype was divided into Luminal A type, Luminal B type, her-2 overexpression type and tri-negative type. Luminal type was ER and PR (progesterone receptor) positive,which requires endocrine therapy. However,adverse reactions were always puzzled patient,for example, hot flashes, osteoporosis, menstrual disorders and other side effects, especially skeletal muscle pain in patients with aromatising inhibitors. Erzhi pills was composed of fructus ligustri lucidi and eclipta, and has the function of calming liver and tonifying kidney Yin.Clinical application was mainly about kidney essence deficiency, medullary sea insufficiency,water failing to nourish wood. As well as,lumbar back ached, lower limb impotence soft,tongue red moss, pulse thin, string thin. Modern pharmacological studies have found that Erzhi pills can antagonize damage and protect liver function of antioxidant action [7]. Erzhi pills has a significant effect on the treatment of kidney diseases caused by liver and kidney Yin deficiency [8,9]. Erzhi pills can stimulate the proliferation of T lymphocytes, thymus cells, regulate vascular endothelial growth factor, matrix metalloprotein 9, and interleukin lL-1, ll-2, lL-12, and other related cytokines which play a role in anti-tumor and immuneenhancing biological processes in vivo [10].It was found that Erzhi pills could improve postoperative endocrine disorders in breast cancer patients [11], and inhibited osteoporosis in postmenopausal women [12]. Domestic experts found that Erzhi pills combined with Guizhi decoction could improve hot flushes in endocrine therapy of Luminal patients [13].Musculoskeletal pain was one of the endocrine side effects affecting the life of breast cancer, and the major factor caused treatment interrupted.Studies had reported that when patients with ER positive received anastrozole and tamoxifen alone or combination, about 28% of them experienced musculoskeletal pain, and 10% of them chose to interrupt treatment because they could not tolerate the adverse reaction [14].Studies had shown that musculoskeletal pain during endocrine therapy was related to the increase in relevant inflammatory cytokines[15,16]. Therefore, this study aimed to explore the regulation and anti-inflammatory mechanism of Erzhi pills on related inflammatory factors in breast cancer cells MCF-7, at the same time,detected the anti-tumor effect on MCF-7, and to provide new ideas for clinical diagnosis and treatment of breast cancer.

In the field of oncology, many experiments had reported that Erzhi pills could induce apoptosis, inhibit the growth and proliferation of tumor cells. Erzhi pills could inhibit the proliferation of HCT116 cells in human colon cancer, induce apoptosis, change the distribution of cell cycles, increase the expression of Bax protein, and reduce the expression of bcl-2 protein [17]. Shang et al reported that Erzhi pills could prolong the average incubation period of breast tumors in rats, reduce the average tumor diameter, volume and tumor weight, and reduce the expression of VEGF and MMP-9 in breast cancer tissues, consequently, inhibit the progression of breast cancer [18]. The results of our study suggested that Erzhi pills could inhibit the growth and proliferation ability of MCF-7 cells, which was consistent with the results reported above.

Wu et al had shown that IL-1β could trigger synovial response, leading to joint damage and pain symptoms [19]. IL-6 was a pleiotropic pro-inflammatory cytokine, and its increased expression was associated with synovitis and bone destruction [20]. IL-18 was an inflammatory factor, also known asγ-interferon inducible factor, which was involved in the biological process of a variety of diseases in the body, such as nephropathy and diabetes. NFκB played an important role in many biological processes such as inflammation, immune regulation, cell proliferation, cell differentiation and cell survival [21]. Studies had shown that NF-κB was an important factor of the pathogenesis of many tumors and might be a potential therapeutic target for breast cancer [22].Moreover, researches had also reported that NFκB was related to inflammation, which might promote the development of breast cancer [23].NF-κB signaling pathway played an important role in inflammatory responses, although the regulate mechanism of NF-κB transcription factors was still unclear, the activation of NFκB transcription factors was regulated by IκBα. In this study, we used high and medium concentrations of drug serum containing Erzhi pills acted on the breast cancer cells MCF-7, then Western blot was applied to detect the protein expressions of IKKα、IκBαand p- NF-κB , which all deregulated, in the NFκB signaling pathway. Meanwhile, mRNA expression of pro-inflammatory factors IL-1β,IL-18 and IL-6 were decreased by RT-PCR,suggesting that Erzhi pills could improve the progression of breast cancer cells MCF-7 by inhibiting inflammatory response.In conclusion, medium and high concentrations of drug serum containing Erzhi pills could suppress the growth and proliferation of breast cancer cells MCF-7 and deregulate the expression of inflammatory factors such as IL-1β, IL-18, IL-6 and the mechanism was related to NF-κB signaling pathway. Erzhi pills may be a potential target for breast cancer treatment and may relieve the adverse reactions such as skeletal muscle pain during endocrine therapy of hormone-receptor positive breast cancer by regulating the NF-κB signaling pathway. This study further confirmed the important role of traditional Chinese medicine in tumor treatment,and showed the unique advantages of traditional Chinese medicine in treatment-related adverse reactions, then improved the quality of life,increased treatment compliance and survival,reduced the possibility of recurrence and metastasis.