Angiotensin-(1-7) [Ang-(1-7)] is a heptapeptide of the renin-angiotensin system with vasodilator and anti-proliferative properties. In the present study, we aim to investigate whether Ang-(1-7) induces apoptosis in breast cancer cells and whether the altered expression of apoptosis-related genes is involved in this process. Human breast cell line T47D was treated with angiotensin-(1-7) and angiotensin II (Ang II). Cell proliferation and apoptosis were quantified using hemocytometer and flow cytometry, respectively. The expression of 84 apoptosis-related genes was evaluated through qPCR array. Ang-(1-7), as opposed to Ang II, decreased proliferation and increased apoptosis in T47D cells. Moreover, many pro-apoptotic genes were up-regulated, such as <i> BAK </i> 1, <i> BAX </i> , <i> BCL </i> 2 <i> L </i> 1, <i> BID </i> and <i> BIK </i> . In addition, some anti-apoptotic genes as <i> AKT </i> 1 and <i> XIAP </i> were down-regulated by heptapeptide. Although a deeper study should be performed, our results support the hypothesis that Ang-(1-7) could change the expression of several genes related to apoptosis, interfering directly in the molecular pathways associated with the survival of breast cancer cells.
Breast cancer is the most common cancer in women and the second leading cause of cancer death in women worldwide [
Studies have shown that the components of the renin-angiotensin system (RAS) act in the carcinogenic process [
The relationship between genetic polymorphisms of the Angiotensin II, Angiotensin-(1-7) and Angiotensin- Converting Enzyme (ACE) with breast cancer was demonstrated in many works [
Programmed cell death (apoptosis) is considered an innate defense mechanism leading the cell to become antineoplastic; similarly, many chemotherapeutic agents act by inducing this type of cell death [
The human ductal breast epithelial tumor cell line T47D was originally obtained from ATCC (American Type Culture Collection). T47D cells was grown in DMEM supplemented with 10% FBS, 100 UI/mL penicillin, 100 ug/mL streptomycin (all from Invitrogen), 2 UI/mL human insulin, and 4 mM glutamine (both from Sigma- Aldrich). The control group was treated with DMEM supplemented only. The experimental groups were treated with DMEM supplemented and peptides: Ang II and Ang-(1-7) in the concentration of 10−6 M. The peptides were replaced daily due to their rapid degradation [
T47D cells were seeded into 24-well plates (1 × 103 cells/well). After 2, 6, 9, and 15 days the cells were removed from triplicate wells using trypsin/EDTA and counted using a hemocytometer.
T47D cells were seeded into 24-well plates. After 2, 6, 9, and 15 days of treatment the cells were removed from triplicate wells using trypsin/EDTA. Posteriorly, cells were stained with Annexin V using the Guava Nexin kit (Millipore) according to the manufacturer’s instructions. The cells from each well were resuspended in 100 µl of supplemented DMEM (1 × 105 cells/well) and 100 µl of the Guava Nexin Reagent. Samples were incubated in the dark at room temperature for 20 min and subsequently evaluated in the Guava easyCyte™ cytometer (Millipore).
Total RNA was extracted from T47D cells (treated with peptides for 48 hours) using the TRIzol reagent (Invitrogen), according to the manufacturer’s instructions, and subjected to DNase treatment using an RNase-free DNase kit (Qiagen). The quantity and quality of extracted RNA were measured by spectrophotometer (NanoDrop Technologies).
The Human Apoptosis RT2 Profiler PCR Array (SA Bioscience) was used to simultaneously evaluate the expression profile of 84 key genes involved in programmed cell death.
Total RNA (2 μg) was converted to cDNA via reverse transcriptase reaction using the First Strand Kit (SABiosciences), according to the manufacturer’s instructions. This kit includes a proprietary buffer that eliminates residual genomic DNA contamination in cell line RNA samples. A reaction mix containing cDNA and all the optimized reagents and buffers needed for SYBR® Green were prepared based upon real-time polymerase chain reactions. Twenty-five microliters of this mix was added to each well of the 96-well plate, which was then loaded out on a 7500 Fast Real-Time PCR System (Applied Biosystems) and run in a two-step cycling program with one initial cycle of 10 min at 95˚C and 40 cycles of 15 s at 95˚C, and one final cycle of 1 min at 60˚C, in a 7500 Fast Real-Time PCR System (Applied Biosystems).
Cell proliferation and apoptosis assays were analyzed by descriptive statistics (means and standard deviation) and inferential statistics through the Student’s t-test with significance level of 5% (p < 0.05). qPCR Array reactions were analyzed trough the RT2 Profiler™ PCR Array Data Analysis software (SABiosciences). We only considered p values below 0.05 and fold regulation values higher than 2.00 to be statistically significant.
Ang-(1-7) decreases the proliferation of T47D cells, while Ang II increased the proliferation of these cells (
Ang-(1-7) increased apoptosis in T47D cells after 6, 9 and 15 days (
The qPCR Array assay was performed to determine the expression profiles of 84 apoptosis-related genes. Ang- (1-7) altered the expression of 60 genes compared to the control group; 47 were up-regulated and 13 were down- regulated, whereas Ang II altered the expression of 22 genes; 7 were up-regulated and 15 were down-regulated (
Some anti-apoptotic genes were down-regulated due Ang-(1-7) treatment (
In the present study we investigated the pro-apoptotic effects of the Ang-(1-7) in human ductal breast epithelial tumor cell line T47D. This line was selected specially for display luminal phenotype with positive expression of estrogen and progesterone, thus constitutes an experimental model which represents approximately 75% of cases of breast cancer diagnosed in clinical practice.
Our results show that Ang-(1-7) hormone has anti-proliferative and pro-apoptotic actions on T47D breast cancer cells. A similar effect was evidenced in studies with human prostate malignant cells [
Ang-(1-7) inhibited the proliferation of T47D breast cancer cells, whereas Ang II favored the proliferation of these cells. Other studies conducted in mammary cells showed similar results [
Analysis of apoptosis by flow cytometry showed that Ang-(1-7) is able to induce apoptosis in breast tumor cell line, but does not cause this effect in normal mammary epithelial cell line MCF-10A. In addition, the qPCR assay performed in T47D cells demonstrated that the Ang-(1-7) might change the expression of more than 70% of apoptosis-related genes (55.9% genes were up-regulated and 15.5% were down-regulated). On the other hand, Ang II changed the expression of 26% of apoptosis-related genes (8.3% genes up-regulated and 17.9% genes down-regulated).
Apoptosis is induced via two main routes involving either the mitochondria (the intrinsic pathway) or the activation of death receptors (the extrinsic pathway). The intrinsic pathway is induced by various types of intracellular stress and is mediated by members of the BCL-2 family. The extrinsic signaling pathway involves the activation of transmembrane death receptors that are members of the TNF superfamily. Both pathways converge to induce the activation of initiator and executioner caspases, leading in programmed cell death [
The expression of caspases 1, 2, 4, 5, 7, 9, 10 and 14 was increased after treatment with Ang-(1-7). The association of these caspases with apoptosis has been described in several studies [
Gene | Fold change | Fold change | Gene | Fold change | Fold change |
---|---|---|---|---|---|
Symbol | Ang-(1-7) × CT | Ang-II × CT | Symbol | Ang-(1-7) × CT | Ang II × CT |
ABL1 | 14.5827 | 0.8335 | CASP7 | 3.5602 | 0.831 |
AKT1 | 0.4335 | 0.422 | CASP8 | 0.5605 | 0.8348 |
APAF1 | 1.2685 | 0.836 | CASP9 | 3.8289 | 1.6668 |
BAD | 1.6643 | 0.8292 | CD40 | 2.9917 | 2.0927 |
BAG1 | 1.2078 | 0.8423 | CD40LG | 9.5977 | 0.8327 |
BAG3 | 0.8741 | 0.8415 | CFLAR | 5.4723 | 1.7056 |
BAG4 | 0.2102 | 0.8399 | CIDEA | 9.5977 | 3.5808 |
BAK1 | 2.604 | 1.6774 | CIDEB | 1.4202 | 0.4142 |
BAX | 9.3295 | 0.4154 | CRADD | 0.9825 | 1.6924 |
BCL10 | 0.1657 | 1.6664 | DAPK1 | 9.5977 | 0.8327 |
BCL2 | 14.7275 | 1.6287 | DFFA | 0.1422 | 0.8319 |
BCL2A1 | 0.2139 | 1.6847 | FADD | 0.4021 | 0.4185 |
BCL2L1 | 10.5406 | 0.4134 | FAS | 0.3109 | 0.4174 |
BCL2L10 | 9.5977 | 0.8493 | FASLG | 9.5977 | 0.8327 |
BCL2L11 | 0.8152 | 1.6665 | GADD45A | 2.2975 | 0.4233 |
BCL2L2 | 1.1388 | 1.6713 | HRK | 0.9564 | 1.6665 |
BCLAF1 | 0.1145 | 1.6424 | IGF1R | 1.4168 | 3.3446 |
BFAR | 0.1481 | 1.6861 | LTA | 9.5977 | 0.8879 |
BID | 2.2737 | 0.8362 | LTBR | 9.4426 | 0.8435 |
BIK | 4.5455 | 1.3173 | MCL1 | 10.1351 | 3.3414 |
NAIP | 2.345 | 0.8453 | NOL3 | 0.7898 | 1.6754 |
BIRC2 | 0.8614 | 1.6483 | PYCARD | 3.2496 | 0.8455 |
BIRC3 | 0.8803 | 0.8456 | RIPK2 | 3.0304 | 1.6477 |
XIAP | 0.2851 | 0.8333 | TNF | 19.5397 | 3.3521 |
BIRC6 | 2.862 | 1.69 | TNFRSF10A | 0.9505 | 0.2116 |
BIRC8 | 9.5977 | 3.5536 | TNFRSF10B | 0.6384 | 0.8365 |
BNIP1 | 0.3422 | 0.8315 | TNFRSF11B | 9.5977 | 0.8327 |
BNIP2 | 3.2399 | 0.8434 | TNFRSF1A | 5.4577 | 1.6898 |
BNIP3 | 0.6969 | 1.6738 | TNFRSF21 | 1.0109 | 0.8427 |
BNIP3L | 1.8115 | 1.6647 | TNFRSF25 | 1.1189 | 0.4218 |
BRAF | 2.5251 | 1.6606 | CD27 | 9.048 | 1.0456 |
NOD1 | 2.4816 | 1.6586 | TNFRSF9 | 9.5977 | 0.8708 |
CARD6 | 1.115 | 0.4216 | TNFSF10 | 5.1556 | 0.8337 |
CARD8 | 0.5609 | 1.0618 | CD70 | 9.5977 | 2.8259 |
CASP1 | 2.3419 | 1.6759 | TNFSF8 | 9.5977 | 0.8327 |
CASP10 | 9.5977 | 0.8582 | TP53 | 0.2649 | 0.6586 |
CASP14 | 8.9969 | 0.7806 | TP53BP2 | 0.1871 | 0.8422 |
CASP2 | 7.9715 | 0.4181 | TP73 | 2.2379 | 1.6741 |
CASP3 | 1.1176 | 1.6624 | TRADD | 2.2328 | 0.8334 |
CASP4 | 5.3219 | 0.4193 | TRAF2 | 3.0765 | 0.42 |
CASP5 | 9.5977 | 0.8327 | TRAF3 | 7.3658 | 0.8305 |
CASP6 | 1.7785 | 0.4178 | TRAF4 | 3.6426 | 0.2059 |
Fold-change and fold-regulation values greater than 2 are indicated in red, fold-change values smaller than 0.05, and fold-regulation values smaller than −2 are indicated in blue.
Pro-apoptotic genes of the BCL-2 family, such as BAX, BID, BIK and BAK, were up-regulated after treatment with Ang-(1-7). The induction of apoptosis by these genes has been fully demonstrated in several studies [
Estrogen-dependent cancers, such as breast and endometrial cancer, exhibit high expression of the BCL-2 gene (anti-apoptotic) and possibly for this reason, have low level of apoptosis [
BIK has the capacity to induce apoptosis in mammary adenocarcinoma cells [
The genes of the TNF superfamily, including TNF, TNFSF10, TNFSF8 and TNFRSF11B, encode apoptotic proteins [
Ang-(1-7) induced the expression of CD40 and CD40L genes. Studies conducted in pancreatic cancer cell lines demonstrated that the expression of these genes have growth inhibitory effect [
CFLAR, CIDEA, DAPK1, FASLG, CD27 and CD70 genes induce cell death [
The TP53 gene, encoding the p53 protein, is mutated in line T47D and perhaps for this reason was not observed increased expression of this gene suppressor tumor after treatment with the Ang-(1-7). However, there was a significant increase in expression of TP73, encoding a protein of very similar role to TP53. The TP73 gene is capable of acting on BAX, BCL2L1, MCL and Caspase 9 [
In this study, some genes considered anti-apoptotic or apoptosis suppressing, such as AKT1, BAG4, BCL2A1, BCLAF1, BFAR, FADD, FAS and XIAP [
The AKT1 gene encodes the Protein Kinase B, which is capable of inhibiting the protein BAD through phosphorylation and induce anti-apoptotic effects [
The XIAP gene protects endometrium carcinoma cells against various pro-apoptotic agents [
In summary, apoptosis was increased and the expression of several genes related to apoptosis was changed after Ang-(1-7) treatment.
Our results support the hypothesis that Ang-(1-7) could change the expression of several apoptosis-related genes, interfering directly in the molecular pathways associated with the survival of breast cancer cells. In vivo assays are being conducted to confirm the potential of this hormone as an antitumor agent.
The work was supported by grants number 2008/54383-0, 2010/03658-9 and 2011/08531-0 from the Sao Paulo Research Foundation (FAPESP)-Brazil.
ACE: Angiotensin-Converting Enzyme;
Ang II: Angiotensin II;
Ang-(1-7): Angiotensin-(1-7);
cDNA: Complementary DNA;
DMEM: Dulbecco’s Modified Eagle Medium;
FBS: Fetal Bovine Serum;
PCR: Polymerase Chain Reaction;
qPCR: Real-Time PCR or Quantitative PCR;
RNA: Ribonucleic Acid;
RAS: Renin-Angiotensin System.