Selenium donors inhibits osteoclastogenesis through inhibiting IL-6 and plays a pivotal role in bone metastasis from breast cancer

Abstract

Bone metastases are a frequent complication of breast cancer, and there has been little progress in the treatment of breast cancer patients with bone metastases. The cytotoxicity of selenium donors, including organic selenium and selenium nanoparticles (SeNPs), to cancer cells has been reported previously, but their relationship with bone metastases progression is not fully clear yet. In this study, multicenter clinical exploration was conducted to obtain dietary selenium intakes of breast cancer patients with or without bone metastasis, to study the relationship between selenium and breast cancer prognosis and bone metastasis. We found that dietary selenium intakes were significantly lower in breast cancer patients with bone metastasis, comparing with the non-bone metastasis cases. Selenium lower group of bone metastasis breast cancer patients had worse prognosis, whereas the daily selenium intakes could not predict the prognosis of breast cancer patients without bone metastasis. Subsequently, we study the regulatory role of selenium donors on bone metastasis at the cellular level, by challenging the cells with SeNPs. SeNPs showed potent cytotoxicity in breast cancer cells, no matter whether they were primary or bone-metastatic. SeNPs treated cancer cell inhibited the survival and differentiation of osteoclast progenitor cells. At the molecular level, we demonstrated that IL-6 partially mediated osteoclastogenesis suppression by SeNPs. These results provide a new way for biomarkers or drug development to treat and even prevent bone metastases of breast cancer by using selenium donors.

INTRODUCTION

Breast cancer is one of the most common malignancies in women worldwide [1]. The majority of breast cancer patients have no evidence of metastatic disease at the time of diagnosis, yet ~ 30% of patients experience recurrent breast cancer in the form of metastasis, of which the most prominent site is bone [2]. Bone metastases are a frequent complication of cancer, the survival time of these patients was very short and >60% of patients will die within 6 months [3]. Unfortunately, there has been little progress in the treatment of breast cancer patients with bone metastases.

Laboratory and animal studies suggest beneficial effects against cancers and little side effects of some dietary microelements supplements, including zinc [4], selenium [5], iron [6] and copper [7]. Especially, selenium supplements have been used in individualized anticancer therapies [8]. Recently, it has been found that the synthesized nanoparticles containing selenium [selenium nanoparticles (SeNPs)], a novel type of selenium donors, have stronger antitumor activity and lower toxicity than their natural forms [9–11]. However, little is known about the functions of selenium donors, including SeNPs and natural selenium, on tumor bone metastasis.

Disseminated tumor cells (DTCs) are frequently detected in bone marrow aspirates of breast cancer patients, regardless of breast cancer subtype and even in those who have early-stage disease and are predictive of decreased recurrence-free survival [12]. These and other such findings support the idea that DTCs find a hospitable niche in the bone marrow. Bone metastatic niches in which DTCs reside have been defined as microdomains within the bone that support tumor cell seeding and outgrowth and are predominantly comprised of hematopoietic cells, mesenchymal stromal cells, osteoblasts, osteoclasts and/or vascular cells [13]. Paracrine interactions between DTCs and these various stromal cells disrupt bone homeostasis to fuel metastatic progression. It is well established that DTCs secrete a variety of cytokines that promote osteoclast activity, which, in turn, causes the release of a variety of tumor-promoting growth factors from the bone, thus propagating a vicious cycle of tumor outgrowth and osteolytic bone breakdown, and this is considered as one of the important mechanisms of bone metastasis [14]. In the clinical research level of this study, we found that patients with higher selenium intakes had lower bone metastasis risk and better prognosis by means of multicenter exploring. At the cellular level, we found that selenium donors could inhibit the survival and differentiation of osteoclast progenitor cells by IL-6 down-regulation and therefore block the bone metastasis in breast cancers.

MATERIALS AND METHODS

Patients

A total of 75 breast cancer patients with bone metastases and 80 cases of age matched primary breast cancer patients without bone metastases were consecutively recruited from four regional hospitals in China, including Binzhou People’s Hospital (64 cases), Zibo Central Hospital (42 cases), Chongqing University central Hospital (14 cases) and Binzhou Medical University Hospital (35 cases). Clinical information was collected, including age, gestational phase, clinical stage, tumor size, histological diagnosis, therapeutic approach and follow-up information. Individualized therapy was performed for each patient. The strategy was designed according to the tumor stage and tumor size of the patient. The putative therapeutic approaches and the corresponding outcomes were all clarified with the patients, and the therapeutic timing and approach were agreed and decided by both patients and doctors. The presence of bone or extraskeletal metastases was determined by biopsy or imaging including bone scans, fluorodeoxyglucose-positron emission tomography, computed tomography and magnetic resonance imaging. Radiologically, abnormal lesions without pathological confirmation found in patients diagnosed with other types of invasive cancer within the previous 5 years were not considered as metastatic tumors. The metastatic status of bone lesions that appeared to be in a gray zone was determined by specialists in orthopedic oncology. Dietary intakes data were assessed by using a 24-h recall survey according to the previous report [15]. The dietary intake data were used to estimate the types and amounts of foods and beverages consumed during the 24-h period before the interview. Daily aggregates of food energy, nutrients and other food components from all foods and beverages were calculated using US Department of Agriculture Food and Nutrient Database for Dietary Studies. The dietary supplements were obtained during the household interview. The total daily selenium intake was calculated by summing each participant’s selenium intake from diet and the average daily amount from supplements.

Preparation of SeNPs

SeNPs was kindly provided by Dr Di Wang, and the physicochemical characteristics were described in their previous publication [10]. In the process of their synthesizing this SeNPs, a gradual color change of the solution with time from initial pale yellow to red color was observed, and no further color change was identified after 48 h of incubation time. The excitation of surface plasmon vibrations of the SeNPs has instigated the final red color to the sample. The average crystalline size of this kind of SeNPs was calculated to be 88.89 nm by X-ray diffraction, and it was identified as a hexagonal ring structure with a pattern of diffraction rings [10]. The energy dispersive X-ray spectrum (EDS) spectrum of the biosynthesized SeNPs has presented an intense peak at 1.5 keV signifying the entire composition of Se in the developed SeNPs. In addition, few weak signals of Cu, P, Cl, Si and Na were also recorded possibly related with the glass underlay. The C, P and O signals were associated with either capped or closely pre-sent biomolecules like enzymes, proteins or bacterial biomaterial [10].

Cell culture

Human breast cancer cell lines (MDA-MB-231 and MCF7) as well as RAW264.7 were obtained from American type culture collection (ATCC). Bone marrow-derived macrophages (BMDMs) were isolated from C57/BL6 mice as described previously, and the cells were cultured in the complete DMEM media supplemented with granulocyte-macrophage colony stimulating factor (GM-CSF) (10 ng/ml). RAW264.7, MCF7 and MDA-MB-231 cells were cultured in DMEM (Gibco, USA) supplemented with 10% fatal bovine serum (FBS) (Hyclone, USA) and 0.01 mg/ml human recombinant insulin (Sigma, USA).

MTT assay

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (C0009, Beyotime, Shanghai, China) was used to determine the cell viabilities. Each well of 96-well plate was seeded with ~10 000 cells and incubated for 24 h. Later, cells are processed at a specific time. Finally, MTT solution was used to treat the cells and incubated for 3 h followed by the addition of dimethyl sulfoxid (DMSO) and rested for 15 min. Enzyme-linked immunosorbent assay (ELISA) microplate reader (DYNEX, USA) was used to measure the absorbance at 570 nm (optical density (OD) value).

Collection of conditioned media

MDA-MB-231 and MCF7 cells were treated with or without low dose of SeNPs (25 μg/ml). Twenty-four hours after treatment, the media from MDA-MB-231 and MCF7 cells were collected as conditioned media.

Proliferation and differentiation of BMDMs

BMDMs (5000 cells/well) were seeded into 96-well plate and incubated with M-CSF (10 ng/ml) and the receptor activator of nuclear factor-κB ligand (RANKL) (50 ng/ml) in the presence of 30% conditioned media, which was replaced every 48 h. The proliferation of BMDM cells was analyzed using MTT assay. After 5–7 days, BMDMs differentiation was analyzed by tartrate-resistant acid phosphatase (TRAP) staining and actin ring formation assays [16].

Animal models

To explore if-selenium donors can prevent bone metastasis, the left ventricle of nude mice were inoculated with luciferase-labeled MDA-MB-231 cells and then given intraperitoneal injection of 20%/80% DMSO/saline or 100 mg/single SeNPs per day for up to 3 weeks. Bone metastases were analyzed by X-ray and histopathologically confirmed with H&E staining. All experimental mice were monitored with an ex vivo imaging system (IVIS200, Caliper LS, Hopkinton, MA, USA).

Reverse transcription-quantitative polymerase chain reaction assay

Total RNA was extracted from cells using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Media without cells served as a negative control for this experiment. Subsequently, reverse transcription-quantitative polymerase chain reaction was carried out with the PrimeScript reverse transcription-polymerase chain reaction (RT-PCR) kit (Takara, Bio, Inc., Shiga, Japan), using β-actin as an internal control, in the Eppendorf Realplex4 machine (cat. no. X222687G; Hamburg, Germany). Reverse transcription reactions were performed using the following parameters: 16°C for 30 min, 42°C for 30 min and 84°C for 5 min. The 2^−∆∆Cq method was used for normalization.

Enzyme-linked immunosorbent assay

The pro-inflammatory cytokines in the media were detected using ELISA. The ELISA kits used here were all obtained from R&D (Minneapolis, MN, USA). Specific experimental steps and data analysis methods were performed as per the manufacturer’s protocols.

Statistical analysis

The results are expressed as mean ± standard deviation (SD) from at least three separate experiments performed in triplicate. The differences between groups were determined using two-tailed Student’s t-test, using SPSS software (Armonk, NY, USA). P values of <0.05 were considered significant. The Chi-square test or Fisher’s exact test was used to analyze the relationship between daily total selenium intakes and the clinicopathological features of the patients. Log-rank test was used for univariate analysis, and Cox regression model was used for multivariate analysis.

RESULTS

Daily total selenium intakes in breast cancer patients with and without bone metastasis

Multicentre clinical experiments were first conducted in four tertiary hospitals to determine the relationship between daily total selenium intakes and breast cancer progressions. The detailed information of the included breast cancer patients with and without bone metastasis are summarized in Table 1. There were no significant differences between bone metastasis and non-bone metastasis group in age, tumor size and tumor stage. In this case, the daily selenium in bone metastasis group was significantly lower than those in non-bone metastasis cases (Table 1).

Table 1.

Characteristics of breast cancer patients

Characteristics	Bone metastases (n = 75)		Non-bone metastases (n = 80)		P
	Number	%	Number	%
Characteristics (chi-square tests)
Age
≤55	40	53.3	49	61.3	0.356
>55	35	46.7	31	38.7
Tumor size
≤5 cm	30	40.0	42	52.5	0.119
>5 cm	45	60.0	38	47.5
Tumor stage
I or II	33	44.0	45	56.3	0.127
III or IV	42	56.0	35	43.7
Daily selenium intake (mcg) (mean ± SD, Student’s t-test)	89.7 ± 43.4		119.5 ± 49.7		0.001

Daily total selenium intakes were associated with the prognosis in breast cancer patients with bone metastasis

Subsequently, we further determine whether daily total selenium intakes have clinical significance as a prognostic marker in breast cancer patients with or without bone metastasis. The mean daily total selenium (mean value = 119.5 mcg) intakes were used as a cutoff value to divide the 80 non-bone metastasis cases into two groups (selenium high vs selenium low) first. Data indicated that there were no significant differences between selenium high and selenium low group in age, tumor size, tumor stage (Table 2) and overall survival (OS) prognosis (Fig. 1A). Next, similar clinical significance of the selenium intakes was also determined in the 75 bone metastasis breast cancer patients with the mean daily total selenium intakes as the cutoff (mean value = 89.7 mcg). The patients in selenium high group have significant better OS prognosis (Fig. 1B), together with smaller tumor size and less late stage (Table 2). Univariate analyses of clinical variables considered potential predictors of mortality in bone metastasis breast cancer patients are shown in Table 3. By log-rank analyses, daily total selenium intakes patterns, age, stage classification and tumor size were identified as potential predictors of mortality in bone metastasis patients. Multivariate analysis was shown in Table 4. Results indicated that tumor stage and selenium intakes were independent factors that affect the mortality. Patients with stage III or IV had a higher mortality risk than those with stage I or II, with the hazard ratio (HR) value at 1.822 [95% (confidence interval) CI: 1.027–3.231]. Patients in selenium higher group had significant lower mortality risk than selenium lower group, with HR value at 2.159 (95% CI: 1.298–3.713). All these data indicate that higher daily total selenium intakes patterns were associated with a better prognosis in bone metastasis patients.

Table 2.

Relationship between daily selenium in takes and clinical feature in breast cancer patients with or without bone metastases

Characteristics	Bone metastases		P	Non-bone metastases		P
	Se lower (≤89.7)	Se higher (>89.7)		Se lower (≤119.5)	Se higher (>119.5)
Total patients	37	38		42	38
Age
>55	19	16	0.422	20	11	0.299
≤55	18	22		22	27
Tumor size
≤5 cm	10	20	0.024	19	23	0.171
>5 cm	27	18		23	15
Stage
I or II	10	25	<0.001	20	25	0.102
III or IV	27	13		22	13

Se, selenium.

Chi-square tests were performed to determine the differences between the two groups.

Figure 1.

Daily total selenium intakes were association between daily total selenium intakes with the overall survival prognosis. (A and B) Kaplan–Meier analysis of total selenium intakes was determined in (A) non-bone metastasis breast cancer patients and in (B) bone metastasis breast cancer patients

Table 3.

Prediction of mortality in breast cancer patients with bone metastases: univariate

		Number	Median survival time (weeks)	95% CI	c2	P
Age	≤55	40	38.00	20.54 ~ 55.46	10.177	0.001
	>55	35	19.00	17.31 ~ 20.69
Tumor size	≤5 cm	30	39.00	22.06 ~ 55.94	15.733	0.000
	>5 cm	45	19.00	16.77 ~ 21.23
Tumor stage	I or II	33	34.00	16.68 ~ 51.32	15.123	0.000
	III or IV	42	18.00	13.10 ~ 22.90
Se level	Higher (>89.7)	38	43.00	34.44 ~ 51.57	14.398	0.000
	Lower (≤89.7)	37	18.00	15.80 ~ 20.20

Table 4.

Multivariate Cox regression analyses selenium level for mortality of breast cancer patients with bone metastases

	B	SE	Wald	df	P	HR	95% CI for HR
							Lower	Upper
Age (≤55 vs > 55)	0.457	0.277	2.713	1	0.100	1.579	0.917	2.720
Tumor size (≤5 cm vs > 5 cm)	0.536	0.301	3.172	1	0.075	1.709	0.948	3.083
Tumor stage (I or II vs III or IV)	0.600	0.292	4.210	1	0.040	1.822	1.027	3.231
Se level (lower vs higher)	0.786	0.268	8.597	1	0.003	2.195	1.298	3.713

SE, standard error; df, degrees of freedom; HR, hazard ratio.

Cytotoxicities of SeNPs against the breast cancer cells with bone metastasis

We next determined whether selenium donors play a role in the breast cancer cells with bone metastasis. The way of selenium donors was SeNPs treatment, because it could improve efficiency and reduce toxicity. We found that the SeNPs have potent cytotoxicities in a dose-dependent manner (from 25 to 100 μg/ml) on both early-stage breast cancer cell line (MCF7) and metastatic breast cancer cell line (MDA-MB-231), which was predictable based on the antitumor activity of a reported in literature (Fig. 2A–B). Because 25 μg/ml was the lowest concentration of SeNPs with statistically significant activity, we next explore the regulatory molecular mechanism of 25 μg/ml SeNPs on breast cancer cell line. Meanwhile, low dose of SeNPs (25 μg/ml) could decrease many cytokine productions, such as macrophage colony stimulating factor (MCSF), IL-6, RANKL and MMP2 (Fig. 2C). To investigate the effect of SeNPs on survival of breast cancer cell in the bone microenvironment, luciferase-labeled MDA-MB-231 cells were transplanted into BALB/c nude mice via the intratibia route, and the mice were treated by SeNPs at 100 mg/day. Luciferase signals detected in tibias using ex vivo imaging 5 weeks after injection were significantly lower in mice from SeNPs treatment group than in mice from control group (Fig. 2D). Meanwhile, we detected the cytokine productions in serums of the mice, finding that IL-6 was significant decreased in SeNPs treatment group, whereas TNFα cannot be regulated by SeNPs (Fig. 2E–F).

Figure 2.

SeNPs had potent antitumor activities against breast cancer cells in vitro and in vivo. (A and B) Cell viabilities of (A) MCF7 and (B) MDA-MB-231 were determined after 24 h of SeNPs treatment at dose ranges from 0 to 100 μg/ml. (C) mRNA of certain targets in low dose SeNPs (25 μg/ml) treated or not treated MDA-MB-231 cells were determined by RT-PCR. (D) Nude mice were transplanted with luciferase-labeled MDA-MB-231 cells, and then were treated with SeNPs at 100 mg/day or not for 5 weeks. The luciferase signal in tibias were detected and quantified by bioluminescence. (E and F) The (E) IL-6 and (F) TNFα level in serums of mice described in (C) were determined by ELISA assay

SeNPs inhibits the survival and differentiation of osteoclast progenitor cells

The SeNPs (25 μg/ml) regulated cytokines, MCSF, IL-6, RANKL and MMP2, were reported to enhance the differentiation of osteoclast [17]. Therefore, we next explore whether the SeNPs treated cancer cell could inhibit osteoclastogenesis by regulating differentiation of osteoclast. We cultured BMDMs, using conditioned media from low dose SeNPs treated or not treated MDA-MB-231, finding that cultured with media from low dose SeNPs treated MDA-MB-231 significantly decreased the number of BMDMs (Fig. 3A). Further, after treatment with M-CSF and RANKL, BMDMs cultured in media from low dose SeNPs treated MDA-MB-231 showed fewer cells differentiated into TRAP-positive multinucleated osteoclasts than BMDMs cultured in media from untreated MDA-MB-231 (Fig. 3B). The messenger ribonucleic acid (mRNA) level of several osteoclast differentiation indicators, cathepsin K, NFATc1 and c-fos, were significant decreased in the RAW264.7 cells cultured in media from low dose SeNPs treated MDA-MB-231 (Fig. 3C). Collectively, these results indicated that SeNPs treated breast cancer cells could inhibit the survival and differentiation of osteoclast progenitor cells.

Figure 3.

SeNPs inhibits the osteoclast differentiation in vitro. (A) BMDMs were cultured in conditioned media from low dose SeNPs (25 μg/ml) treated or not treated MDA-MB-231 cells. Surviving cells were counted at 24 h. (B) BMDMs were incubated with macrophage colony stimulating factor (M-CSF) and RANKL in the presence of 30% conditioned media from low dose SeNPs treated or not treated MDA-MB-231 cells for 5–7 days. RANKL-induced mouse BMDM differentiation to osteoclast were detected and quantified by TRAP staining. (C) RT-PCR analysis of cathepsin K, NFATc1 and c-fos mRNA expression in RAW264.7 cells cultured in conditioned media from low dose SeNPs treated or not treated MDA-MB-231 cells

SeNPs inhibits osteoclast differentiation through decreasing IL-6

We found that SeNPs could down-regulate the IL-6 level in breast cancer cells and previous reports have demonstrated that IL-6 plays a key role in regulating the osteoclastogenesis and bone metastasis in breast cancers [18, 19]. Therefore, we next explore whether SeNPs regulate osteoclast differentiation through decreasing IL-6. Here, we found that BMDMs cultured in conditioned media from MDA-MB-231 cells treated with low dose of SeNPs displayed lower proliferation and less differentiation to TRAP-positive multinucleated osteoclasts than those cultured in the control group, whereas recombinant IL-6 treatment substantially reversed these effects (Fig. 4A–B). Further, recombinant IL6 reversed SeNPs mediated decrease of cathepsin K, NFATc1, c-fos and TRAP expression in RAW264.7 cells (Fig. 4C). These findings demonstrate that IL-6 down-regulation contributes to the inhibitory function of SeNPs on osteoclastogenesis.

Figure 4.

SeNPs inhibits the osteoclast differentiation in vitro by decreasing IL-6 level. (A) Recombinant human IL-6 treatment (10 ng/ml) reversed the suppressive effects of conditioned medium from low dose SeNPs treated MDA-MB-231 cells on the BMDMs proliferation. (B) Recombinant human IL-6 treatment (10 ng/ml) reversed the suppressive effects of conditioned media from low dose SeNPs treated MDA-MB-231 cells on the BMDM differentiation to osteoclast. (C) Recombinant human IL-6 treatment (10 ng/ml) reversed the suppressive effects of conditioned medium from low dose SeNPs treated MDA-MB-231 cells on cathepsin K, NFATc1 and c-fos mRNA expression in RAW264.7 cells

DISCUSSIONS

It has been proposed that selenium has cancer preventive properties, due the role of this nutrient as a cofactor of several antioxidant enzymes. Many efforts have been performed in exploring the chemoprotective properties of selenium for specific cancers including cancers of the prostate, lung, pancreas, liver, skin, head and neck, bladder, colon and breast [8–11]. However, the data on the relationship between selenium intake and bone metastasis are still lacking. There is epidemiological evidence that the relationship between selenium and cancer progressions still controversial, and one of the main reason is that the selenium intake of patients in different regions will be significantly different due to the characteristics of the local soil and diet, so it is necessary to carry out multicenter data statistics in multiple regions [20, 21]. In this study, from the monitoring results of four tertiary hospitals in different regions, we found that the daily selenium intakes were significantly lower in bone metastasis breast cancer patients than in the non-bone metastasis cases. Further, we found that patients with higher daily selenium intakes had a smaller tumor size and lower risk of late stage, together with the lower OS rate. In multivariate Cox analysis, selenium remained to be an independent prognostic factor. The significance of this clinical result is, first, this result indicated that we can predict the prognosis of patients according to the selenium intake and also suggests that we can adjust the selenium content in patients diet to improve their condition and quality of life; second, it suggests a close association between selenium and pathogenesis of bone metastasis of breast cancer. Therefore, we used SeNPs, which are a type of selenium donors and considerably more effective than the traditional organic selenium or inorganic selenium, to explore the potential role of selenium in regulating bone metastasis of cancer in the cell model.

Although the study of the relationship between selenium donors and bone metastasis is not clear, some reports have indicated that selenium donors can regulate bone tumor and bone disease, which provides important clues for us to study its mechanism of regulating bone metastasis. Wang et al.’s reports indicated that humans with lower levels of dietary selenium intake have a higher prevalence of osteoporosis in a dose-response manner [21]. Panget al. demonstrated that selenium has a strong antitumor effect on osteosarcoma [22]. Yazıcı et al. observed that zoledronic acid, bevacizumab and dexamethasone-induced apoptosis, mitochondrial oxidative stress, and calcium signaling are decreased in human osteoblast-like cell line by the selenium treatment [23]. These reports, together with our clinical results, suggest that selenium donors may be involved in the regulation of bone microenvironment to block bone metastasis of breast cancers.

Our subsequent cell experiments demonstrated that SeNPs suppressed breast cancer cell survival and inhibited osteoclast differentiation in the bone microenvironment, which confirmed the selenium donors could inhibit bone metastasis of breast cancer. It has been reported that SeNPs can regulate multiple tumor-related signaling pathways. Huang et al. have proved that SeNPs can stimulate autophagy of cancer cells and thus play an anticancer role [24]. Vekariya et al. showed that SeNPs inhibited cell growth and synthesis of DNA, RNA or protein, suggesting that SeNPs may alter the expression of a large number of functional molecules including non-coding RNA [25]. Our findings provide an extension of the known SeNPs related signaling pathways. In this study, we found that SeNPs decrease the IL-6 level in breast cancer cells, thereby regulating osteoclast differentiation.

Agents that inhibit the IL-6 activity or expressions have been widely developed as a potential new drug for various diseases. For example, tocilizumab is a recombinant humanized anti-interleukin-6 receptor (IL-6R) monoclonal antibody that has a main use in the treatment of rheumatoid arthritis, systemic juvenile idiopathic arthritis and polyarticular juvenile idiopathic arthritis [26]. Our results, together with the previous reports, provide a new clue for the antitumor mechanism of selenium donors, and a new way for drug development by targeting IL-6.

Conflict of interest statement

There are no conflicts of interest to declare.

Statement of ethics

This study was approved by the ethics committees of Binzhou People’s Hospital, Zibo Central Hospital, Chongqing University central Hospital and Binzhou Medical University Hospital, and all patients provided informed written consent before study enrollment. All the experiments were carried out according to principles of Helsinki declaration.