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Abstract

Breast cancer is one common female specific malignant tumor and has gradually increased incidence. Matrix metalloproteinase-9 (MMMP-9) and its inhibitor TIMP-1 participate in tumor invasion and metastasis. This study analyzed the effect of various treatment approaches on TIMP-1 and MMP-9 levels in terminal stage breast cancer. Post-op breast cancer patients including chemo-radio therapy group, radio-chemo therapy group and simultaneously chemo- and radio-therapy group were compared for efficacy, along with assays for TIMP-1 and MMP-9 levels for analyzing their correlation with clinical-pathological features of breast cancer. Chemo $+$ radio-therapy group had lower focal recurrence and distal metastasis than the other two groups, plus higher 5-year survival rates ( $p<$ 0.05). After treatment, all patients showed lower serum MMP-9 level, activity and higher TIMP-1 levels than those before treatment ( $p<$ 0.05). Concurrent radio $+$ chemo-therapy group showed lower serum MMP-9 level, activity and higher TIMP-1 levels ( $p<$ 0.05 compared to the other two groups). Serum MMP-9 and TIMP-1 levels after treatment are correlated with patient age, pathological grade, tumor size and lymph node metastasis ( $p<$ 0.05). Simultaneous chemo- and radio-therapy on breast cancer patients after surgery could reduce focal recurrent rate or distal metastasis rate, thus improving 5-year survival rate. MMP-9 and TIMP-1 levels are correlated with age, pathological grade, tumor size and lymph node metastasis of breast cancer patients.

Keywords

Breast cancer treatment efficacy TIMP-1 MMP-9

1. Introduction

China has increased incidence of breast cancer by years, with younger disease onset age, thus severely affecting female health. Survey showed that about 50% breast cancer patients were at pre-menopause stage, and about 60% of patients showed positive expression for estrogen [1]. An investigation for pathogenic factors showed that it was under the combined effects of genetic, hormonal, diet, radiation, and benign breast lesions. Commonly applied treatment approaches for breast cancer include surgery, chemotherapy, radiation, hormonal replacement and targeted treatment [2]. With progression of medicine technology, the treatment for breast cancer has been evolved from single approach to focal combined with systemic treatment including hormonal, radio- and chemo-therapy, which can significantly suppress the incidence of adverse effects such as radiation esophagitis or pulmonary fibrosis [3].

Major reasons for breast cancer related death are uncontrollable growth of tumors and distal metastasis, during which tumor cells must degrade extracellular matrix (ECM) for penetrating tissue barrier [4]. The degradation of ECM becomes the most important, and involves the action of multiple hydrolytic enzymes. The major type of these enzymes is matrix metalloproteinases (MMPs), in which type IV collagenase, or MMP-9, was the most widely studied one [5]. MMPs can degrade ECM to facilitate infiltration of malignant tumor cells into peripheral tissues, whilst tissue inhibitor of MMPs (TIMPs) could suppress the activity of MMPs and inhibit tumor invasion or metastasis [6]. TIMP-1 is a catalytic inhibitor for MMPs and one type of growth factor. Study showed the correlation between over-expression of MMPs and TIMPs and recurrence or metastasis of breast cancer [7]. In current study we recruited post-op breast cancer patients from Nantong Traditional Chinese Medicine Hospital and gave them post-op radio-therapy, post-op chemo-therapy, and post-op chemo plus radiotherapy, whose treatment efficiency was analyzed to test expression of MMP-9 and TIMP-1.

2. Materials and methods

2.1 Patient information

A total of 120 estrogen receptor positive breast cancer patients who received improved radical surgery in Nantong Traditional Chinese Medicine Hospital from January 2012 to January 2017 were recruited. Patients aged between 30 and 70 years (average age $=$ 48.5 $\pm$ 3.2 years), and included 88 cases of invasive cancers and 32 non-invasive ones. TNM staging showed 57, 33 and 30 cases of T1, T2 and T3 phase. All patients received oral hormonal drugs, and were further divided into three groups based on treatment strategy: (1) 40 patients with chemotherapy followed by radiotherapy (aging between 35 and 70 years, average age $=$ 49.8 $\pm$ 3.5 years, including 17, 12 and 10 patients at T1, T2 and T3 phases); (2) 40 patients with radiotherapy followed by chemotherapy (aging between 33 and 69 years, average age $=$ 47.5 $\pm$ 3.1 years, including 20, 10 and 12 cases at phase T1, T2 and T3; and (3) 40 patients received chemo- and radio-therapy simultaneously (aging between 35 and 70 years, average age $=$ 49.2 $\pm$ 2.8 years, including 20, 11 and 8 patients at phase T1, T2 and T3. Another cohort of 40 healthy volunteers was recruited from Nantong Traditional Chinese Medicine Hospital as the control group.

2.1.1 Inclusive criteria

All patients received confirmed diagnosis by pathology, and did not receive novel auxiliary treatment before surgery. Hormonal replacement therapy was performed after surgery. All participants have signed informed consents and were able to accomplish this study.

2.1.2 Exclusive criteria

No severe dysfunction in vital organs including heart, liver and lungs. No complication with mesenchymal tissue or immune disorders. All patients did not receive radio-, chemo-, immune-, cyro- or laser- therapy before surgery. Patients were not complicated with malignant tumors in other organs or psychiatric disorders.

2.2 Reagent and equipment

Anastrozole (Yangzijiang Pharma., Certificate Doc# 000201, China); Tamoxifen (Fudan Fuhua Pharma., Certificate Doc# 031103, China). ELISA kit for MMP 09 and TIMP-1. Ultrapure workstation (Formal, USA). Normal centrifuge (Feige, China).

2.3 Patient treatment approach

All patients received hormonal replacement therapy. For menopausal patients, aromase inhibitor Anastrozole (1 mg/d) was applied, whilst other patients received estrogen receptor antagonist Tamoxifen (20 mg/d) for a 5-year course as stipulated by clinicians.

2.3.1 Chemo-radio therapy group

Patients received standard protocol (500 mg/m ${}^{2}$ Anastrozole $+$ 75 mg/m ${}^{2}$ Tamoxifen $+$ 500 mg/m ${}^{2}$ 5-fluorouracil) for 6 weeks, followed by 2-week rest and radiation (50 Gy in chest wall field, or 30 Gy X-ray on supraclavicular filed plus 20 Gy electron beam for those patients with more than 3 metastatic lesions on pit lymph nodes).

Table 1
Comparison of treatment efficiency

Group	$N$	Focal recurrence ( $n$ , %)	Distal metastasis ( $n$ , %)	5-year survival rate ( $n$ , %)
Chemo-radio	40	9 (22.5)	14 (35)	29 (72.5)
Radio-chemo	40	8 (20)	13 (32.5)	30 (75)
Chemo $+$ radio	40	6 (15) ${}^{*\#}$	10 (25) ${}^{*\#}$	35 (87.5) ${}^{*\#}$

Note: *, $p<$ 0.05 compared to chemo-radio group; #, $p<$ 0.05 compared to radio-chemo group.

2.3.2 Radio-chemo therapy group

Radiation therapy started at 2 weeks post-surgery, followed by 2-week rest and chemotherapy. Dosage or radiation field followed those in the chemo-radio therapy group.

2.3.3 Radio $+$ chemo therapy group

Three-week radiotherapy was performed after surgery, followed by routine radiotherapy and another 3-week chemotherapy. Dosage or radiation field followed those in the chemo-radio therapy group.

2.4 ELISA for blood MMP-9 and TIMP-1 levels in patients

All patients were collected for fasted venous blood samples before and after treatment. Serum was isolated from venous blood and stored in fridge. ELISA was performed to detect serum levels of MMP-9 and TIMP-1. In brief, the number of wells required was calculated. The test kit was incubated at room temperature for 30 min, and standard samples were diluted. At each concentration, five replicated wells were tested. Samples were added for dilution, incubation, rinsing, development and quenching. Absorbance values at 450 nm wavelength were measured by a microplate reader. A liner regression function was plotted to calculate sample concentration.

2.5 Measurement of MMP-9 activity

The MMP-9 activity was measured with a specific MMP-9 monoclonal antibody in combination with an MMP-9 fluorogenic substrate, 5-FAM/QXL™520.

FRET peptide using SensoLyte ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ Plus 520 MMP-9 Assay Kit (catalog number: 72017) (AanSpec Inc., Fremont, CA, USA). Briefly, collected serum was centrifuged at 1000 $\times$ g for 10–15 min at 4 ${}^{\circ}$ C followed by collection of the supernatant. Then, 100 $\mu$ L/well samples, MMP-9 standards, and blank control were added to the microplate coated with monoclonal anti human MMP-9 and incubated at room temperature for 1 h followed by washing four times using washing buffer. After that, APMA (1 mM; AnaSpec, San Jose, CA, USA) was added and incubated at 37 ${}^{\circ}$ C for 2 h to activate MMP-9. The activated samples were assayed for MMP-9 activity using the SensoLyte ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ Plus 520 MMP-9 Assay Kit, according to the manufacturer’s instructions. Duplicate samples were incubated at room temperature for 60 min, followed by reading of fluorescence intensity at Ex/Em $=$ 490/520 nm.

2.6 Observing indexes

All patients received 5-year follow-up for comparing treatment efficacy, including incidence of focal recurrence, distal metastasis, and 5-year survival rate.

Serum levels of MMP-9 and TIMP-1 of patients were compared before and after treatment.

One-way and multi-variant analysis was performed to reveal the correlation between serum MMP-9 and TIMP-1 levels 1 month post-treatment and clinical-pathological features.

2.7 Data processing

SPSS17.0 was used for data processing. Enumeration data were analyzed by chi-square test, and measurement data were processed by analysis of variance (ANOVA) and were presented as mean $\pm$ standard deviation (SD). Multi-variant analysis adopted Logistic regression model. A statistical significance was identified when $p<$ 0.05.

Figure 1.

Serum MMP-9 and TIMP-1 levels in patients before and after treatment. *, $p<$ 0.05 compared to before treatment; #, $p<$ 0.05 compared to chemo-radio group; &, $p<$ 0.05 compared to radio-chemo group; @, $p<$ 0.05 compared to control group.

3. Results

3.1 Comparison of treatment efficacy in patients

Treatment efficacy was compared among all groups of patients, including focal recurrent rate, distal metastasis rate and 5-year survival rate. Results showed that radio $+$ chemo therapy group had lower focal recurrence or distal metastasis than chemo-radio or radio-chemo therapy groups, plus higher 5-year survival rate ( $p<$ 0.05). No significant difference of focal recurrence, distal metastasis or 5-year survival rate was observed between chemo-radio therapy and radio-chemo therapy groups ( $p>$ 0.05, Table 1).

3.2 Levels of MMP-9 and TIMP-1 in patients before and after treatment

Peripheral venous blood was collected before and after treatment to test serum MMP-9 and TIMP-1 levels. Results showed significantly higher serum MMP-9 and TIMP-1 levels in all three treatment groups compare to normal control group, with no significant difference among those three groups ( $p>$ 0.05). After treatment, serum MMP-9 and TIMP-1 levels in all three groups were still higher than control ( $p<$ 0.05). Radio $+$ chemo therapy group had lower serum MMP-9 and higher TIMP-1 levels than chemo-radio or radio-chemo therapy group ( $p<$ 0.05). No significant difference existed in serum MMP-9 and TIMP-1 levels between chem-radio and radio-chemo therapy group ( $p>$ 0.05, Fig. 1). Consistently, the MMP-9 activity in patients before and after treatment showed the similar profile to MMP-9 serum level (Fig. 1).

3.3 Correlation between serum MMP-9 or TIMP-1 levels in patients and clinical pathological features

We further compared the relationship between patient serum MMP-9 levels and clinical pathological features, including age, TNM stage, tumor size and lymph node metastasis. Results showed that post-treatment serum MMP-9 and TIMP-1 levels were correlated with patient age, TNM stage, tumor size and lymph node metastasis ( $p<$ 0.05). In those patients with older than 55 years, TNM stage at or beyond stage III, tumor maximal diameter larger than 5 cm or lymph node metastasis, their serum MMP-9 levels were gradually decreased with elevated TIMP-1 levels ( $p<$ 0.05, Table 2).

3.4 Multi-variant analysis for patient serum MMP-9/TIMP-1 levels and clinical/pathological features

Multi-variant Logistic analysis was performed to analyze factors including age, TNM stage, tumor size, and lymph node metastasis. Results showed that age, TNM stage, tumor size and lymph node metastasis were all independent risk factors (Table 3).

Table 2
Correlation between serum MMP-9, TIMP-1 and clinical-pathological features

Parameters	$N$	MMP-9 (ng/ml)		TIMP-1 (ng/ml)
		Before	After	Before	After
Age
$<$ 55	71	42.34 $\pm$ 0.58	35.22 $\pm$ 3.75	281.03 $\pm$ 2.11	288.23 $\pm$ 0.56
$\geqslant$ 55	49	57.97 $\pm$ 0.37	28.67 $\pm$ 4.12	253.02 $\pm$ 1.05	359.83 $\pm$ 0.32
P		$<$ 0.05	$<$ 0.05	$<$ 0.05	$<$ 0.05
Pathology grade
I	57	41.13 $\pm$ 0.45	48.33 $\pm$ 2.97	291.33 $\pm$ 1.91	293.57 $\pm$ 0.44
II	33	51.87 $\pm$ 0.67	35.74 $\pm$ 3.35	269.77 $\pm$ 2.01	312.31 $\pm$ 0.34
III	30	57.02 $\pm$ 0.34	27.98 $\pm$ 5.05	232.56 $\pm$ 1.23	347.05 $\pm$ 0.13
P		$<$ 0.05	$<$ 0.05	$<$ 0.05	$<$ 0.05
Tumor size
$<$ 2 cm	54	41.24 $\pm$ 0.33	41.52 $\pm$ 2.05	290.13 $\pm$ 3.01	295.24 $\pm$ 0.81
2–5 cm	36	50.69 $\pm$ 0.24	35.24 $\pm$ 4.03	271.49 $\pm$ 2.97	365.24 $\pm$ 0.45
$>$ 5 cm	30	55.91 $\pm$ 0.56	20.51 $\pm$ 3.11	259.96 $\pm$ 4.02	397.25 $\pm$ 0.23
P		$<$ 0.05	$<$ 0.05	$<$ 0.05	$<$ 0.05
Lymph node metastasis
No	85	45.92 $\pm$ 0.13	41.04 $\pm$ 2.21	292.01 $\pm$ 2.06	308.17 $\pm$ 0.91
Yes	35	50.47 $\pm$ 0.75	39.57 $\pm$ 5.01	271.38 $\pm$ 1.89	331.88 $\pm$ 0.15
P		$<$ 0.05	$<$ 0.05	$<$ 0.05	$<$ 0.05

Table 3

Multi-variate analysis of serum MMP-9/TIMP-1 levels and clinical/pathological features

Parameters	MMP-9			TIMP-1
	Regression coefficient	P value	Relative risk factor	Regression coefficient	P value	Relative risk
Age	0.724	0.003	2.003	0.801	0.004	2.228
Pathological grade	1.132	0.002	2.015	1.275	0.001	2.852
Tumor size	1.113	0.002	2.367	1.004	0.001	2.730
Lymph node metastasis	0.748	0.002	2.368	0.952	0.003	2.376

4. Discussion

The incidence of breast cancer was gradually increasing in recent years with aging population, thus severely threatening female health. The advancement of molecular biology has transformed the treatment for breast cancer from single surgery toward combined treatment including radio-, chemo-, endocrine- and targeted therapy [8]. However, data still showed no significant improvement of recurrent or metastatic rate of breast cancer, with high mortality. MMPs is one group of endopeptidase dependent on bivalent ions [9]. TIMPs is one family of endogenous regulator inhibiting MMPs activity [10]. Previous study showed important roles of MMPs and TIMPs in onset and progression malignant tumors [11, 12]. This study recruited post-op breast cancer patients to analyze treatment efficacy of post-op radiotherapy, post-op chemotherapy and post-op chemo $+$ radiotherapy, along with levels of MMP-9 and TIMP-1.

In this study, all recruited breast cancer patients were divided based on treatment approaches, and follow-ups were performed for comparing treatment efficiency. Radio $+$ chemotherapy group had lower focal recurrent and distal metastatic rates than chemo-radiotherapy or radio-chemotherapy group, plus higher 5-year survival rate. No significant difference existed in those parameters between chemo-radiotherapy or radio-chemotherapy group. These results showed that for patients with improved radical surgery or retained surgery, simultaneous radio and chemo-therapy after surgery could help to reduce focal recurrent rate and distal metastatic rate, and improved 5-year survival rate, with better efficiency than pure post-op chemo- or radiotherapy. Study showed that for those breast cancer patients with positive expression of estrogen and progesterone receptor and indicators for radiotherapy, hormonal replacement and radiotherapy are critical for improving patient prognosis, although systemic treatment cannot be neglected. Previous studies showed that synchronous radio- and chemo-therapy could obtain better efficacy, and comparable adverse effects with pure chemo- or radio-therapy [13, 14].

In malignant tumors, cytokines for mediating cell proliferation and apoptosis are critical, as their abnormal expression can induce tumor invasion or metastasis. In this study, we measured serum MMP-9 and TIMP-1 levels in patients before and after treatment. No significant difference existed in these parameters in all three groups before treatment. After treatment, all patients had lower serum MMP-9 levels and higher TIMP-1 levels than those before treatment. Chemo $+$ radio-therapy treatment group had further lower serum MMP-9 and higher TIMP-1 than other two pure chemo- or radio-therapy groups.

MMP-9 is secreted out of cells in the enzymogen form to degrade type IV collagen, fibronectin, and affect cell adhesion potency, thus participating tumor cell growth, infiltration and metastasis [15, 16]. TIMPs are distributed in tissues and body fluids. As tissue inhibitor for MMPs, TIMPs can specifically bind with catalytic domain of MMP-9 enzymogen or activated enzymes to form tissue complex, for exerting specifically inhibitory functions on MMP-9 activity [17, 18]. This study showed that synchronous chemo- and radio-therapy in post-op breast cancer patients significantly suppressed patient MMP-9 level whilst increased TIMP-1 level.

This study further analyzed the correlation between patient serum MMP-9 and TIMP-1 levels and clinical/pathological features. Post-treatment serum MMP-9 and TIMP-1 levels were correlated with patient age, TNM stage, tumor size and lymph node metastasis. In those patients with older than 55 years, TNM stage at or beyond stage III, tumor maximal diameter larger than 5 cm or lymph node metastasis, serum MMP-9 levels were gradually decreased, with increased TIMP-1 level. Multi-variate analysis showed that age, TNM stage, tumor size and lymph node metastasis were all independent risk factors affecting serum MMP-9 and TIMP-1 levels. Previous study showed that both MMPs and TIMPs had abnormal expression in various malignant tumors including colon cancer, gastric carcinoma and pancreatic cancer, and were involved in tumor metastasis and invasion [19]. Breast cancer is one malignant tumor with epithelium origin, with potent invasion and metastasis ability. The degradation of matrix in basal membrane is closely correlated with MMPs activity and TIMPs levels at various subtypes [20]. Previous study showed elevated expression of MMP-9 and TIMP-1 in breast cancer tissues, and more potent increase with tumor growth and lymph node metastasis [21]. This study indicated that simultaneous radio- and chemo-therapy on post-op breast cancer patients could decrease serum MMP-9 level, increase TIMP-1 level, and suppress the probability of ECM recurrence, thus suppressing the incidence of tumor focal recurrence or distal metastasis.

5. Conclusion

Simultaneous chemo- and radio-therapy for breast cancer patients after radical resection surgery could decrease focal recurrent or distal metastatic rate, and improved 5-year survival rate. MMP-9 and TIMP-1 levels are correlated with patient age, TNM stage, tumor size and lymph node metastasis. Simultaneous chemo- and radio-therapy after surgery could help to decrease MMP-9 and increase TIMP-1 levels in breast cancer patients, with better efficacy than pure chemo- or radio-therapy after surgery. MMP-9 and TIMP-1 thus may become indicators predicting prognosis of breast cancer. Future studies can be pursued in cell or animal model to investigate detailed functional mechanism.

Footnotes

Acknowledgments

This work was supported by Nantong City Social Development Fund (No. MS12017017-4).

Conflict of interest

None.

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Effects of various treatment approaches for treatment efficacy for late stage breast cancer and expression level of TIMP-1 and MMP-9

Abstract

Keywords

1. Introduction

2. Materials and methods

2.1 Patient information

2.1.1 Inclusive criteria

2.1.2 Exclusive criteria

2.2 Reagent and equipment

2.3 Patient treatment approach

2.3.1 Chemo-radio therapy group

Table 1 Comparison of treatment efficiency

2.3.3 Radio + chemo therapy group

2.4 ELISA for blood MMP-9 and TIMP-1 levels in patients

2.5 Measurement of MMP-9 activity

2.6 Observing indexes

2.7 Data processing

3.1 Comparison of treatment efficacy in patients

3.2 Levels of MMP-9 and TIMP-1 in patients before and after treatment

3.3 Correlation between serum MMP-9 or TIMP-1 levels in patients and clinical pathological features

3.4 Multi-variant analysis for patient serum MMP-9/TIMP-1 levels and clinical/pathological features

Table 2 Correlation between serum MMP-9, TIMP-1 and clinical-pathological features

5. Conclusion

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
Comparison of treatment efficiency

2.3.3 Radio $+$ chemo therapy group

Table 2
Correlation between serum MMP-9, TIMP-1 and clinical-pathological features