|Year : 2015 | Volume
| Issue : 2 | Page : 345-351
Sensitivity of endometrial cancer cells from primary human tumor samples to new potential anticancer peptide lactaptin
Olga A Koval1, Galiya R Sakaeva2, Alexander S Fomin2, Anna A Nushtaeva2, Dmitry V Semenov2, Elena V Kuligina2, Ludmila F Gulyaeva3, Alexey V Gerasimov4, Vladimir A Richter2
1 Institute of Chemical Biology and Fundamental Medicine SB RAS; Novosibirsk State University, Novosibirsk, Russia
2 Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia
3 Novosibirsk State University; Institute of Molecular Biology and Biophysics, SB RAMS, Novosibirsk, Russia
4 National Novosibirsk Regional Oncology Dispensary, Novosibirsk, Russia
|Date of Web Publication||7-Jul-2015|
Olga A Koval
Institute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentieva 8, Novosibirsk-630 090
Source of Support: The following grants: SB RAS Integrated
Program (project # 94, RFBR # 13-04-01457), Russian Federal Target Program (project with unique identifier RFMEFI60714X0063), Conflict of Interest: None
Purpose: Endometrial carcinoma is the most common gynecologic malignancy which is associated with a poor prognosis when diagnosed at an advanced stage; therefore, the discovery of efficacious new drugs is required to reinforce conventional chemotherapy. Short-term cultures of primary cells from endometrial tumors could be used for testing new anticancer therapeutics as well as for the development of personalized cancer therapy strategy. Here, the antitumor effect of a recombinant analogue of lactaptin (RL2), a new potential anticancer molecule, was examined against primary human endometrial cancer cells.
Materials and Methods: Primary cell cultures of malignant and normal human endometrium were performed by enzymatic digestion of endometrial tissue from biopsy material. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the messenger ribonucleic acid (mRNA) state of estrogen (ERs) and progesterone (PRs) hormone receptors and aromatase (Cyp 19) in cell cultures. Dynamic monitoring of cell adhesion and proliferation was made using the iCELLigence system (ASEA Biosciences). The sensitivity of cell cultures to conventional anticancer drugs and the lactaptin analog was estimated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, flow cytometry, and the iCELLligence system.
Results: Established short-term primary cultures of endometrial cancer cells were ERα/ERβ/PR-positive and sensitive for RL2. The IC 50 values of doxorubicin and cisplatin were determined for all of the primary cultures designed. KE normal cells displaying low Cyp19 mRNA levels and high ERβ and PR mRNA levels were more resistant to RL2 treatment as well as to cisplatin and doxorubicin.
Conclusions: Our results indicate that the recombinant analog of lactaptin, RL2, exerts cytotoxic effects against primary hormone-dependent endometrial tumor cells in vitro with features of apoptosis.
Keywords: Apoptosis, endometrial carcinoma, estrogen receptors, lactaptin, primary cultures
|How to cite this article:|
Koval OA, Sakaeva GR, Fomin AS, Nushtaeva AA, Semenov DV, Kuligina EV, Gulyaeva LF, Gerasimov AV, Richter VA. Sensitivity of endometrial cancer cells from primary human tumor samples to new potential anticancer peptide lactaptin. J Can Res Ther 2015;11:345-51
|How to cite this URL:|
Koval OA, Sakaeva GR, Fomin AS, Nushtaeva AA, Semenov DV, Kuligina EV, Gulyaeva LF, Gerasimov AV, Richter VA. Sensitivity of endometrial cancer cells from primary human tumor samples to new potential anticancer peptide lactaptin. J Can Res Ther [serial online] 2015 [cited 2020 Jan 27];11:345-51. Available from: http://www.cancerjournal.net/text.asp?2015/11/2/345/157301
| > Introduction|| |
Endometrial carcinoma is the most common gynecologic malignancy and is associated with a poor prognosis when diagnosed at an advanced stage. Annually, approximately 64,000 new cases of endometrial cancers are detected in Europe.  Endometrial adenocarcinomas are the third most frequent cases of cancer in woman aged 40-54 years (8.6%) in the Russian Federation; 19,800 new cases of endometrial carcinoma are diagnosed every year.  Endometrial cancers are very heterogeneous and even histologically similar tumors may respond to therapy differently. Therefore, individual tumor cases should be defined by molecular markers to determine the best treatment approach. The status of steroid hormones receptors and molecules functioning in hormones metabolism are the main prognostic markers for endometrial carcinomas. , Response rates to antiestrogen therapy by progestins, tamoxifen, and aromatase inhibitors can be predicted by the presence of progesterone (PR) and estrogen receptors (ERs) in advanced or recurrent endometrial tumors and are approximately 15-20%.  Among the patients with Grade 1 endometrial adenocarcinoma, more than 75% respond to progestin treatment.  Doxorubicin combined with cisplatin without or with taxane has played an important role in the treatment of high-risk disease, but is also associated with infrequent complete response. , To improve the response rate of endometrial tumors and to overcome drug resistance, it is essential to develop new effective second-line cytotoxic agents that increase primary chemotherapy. Tentative screening of individual carcinomas to chemotherapeutic drugs probably improves the treatment outcomes for endometrial cancer in vivo. That personalized approach can be realized using patient-derived primary tumor cultures.
Lactaptin, a fragment of human milk kappa-casein (residues 57-134) was shown to induce apoptosis of MCF-7 adenocarcinoma cells in vitro, while having no effect on the viability of nonmalignant mesenchymal stem cells (MSC) cells.  A recombinant analog of lactaptin containing complete amino acid sequences of lactaptin (RL2) was shown to retain the biological activity of the natural peptide.  The mechanism leading to cell death after RL2 treatment involves penetration of the peptide into cells, disruption of the inner mitochondrial membrane potential, down regulation of BCL-2, activation of the executer caspase-3 and -7, and apoptotic fragmentation of deoxyribonucleic acid (DNA). The tumor suppression efficacy of the recombinant analog of lactaptin in vivo was shown against mouse hepatoma as well as MDA-MB-231 human adenocarcinoma cells grafted onto severe combined immunodeficiency (SCID) mice.  It was demonstrated that the recombinant analog of lactaptin induced apoptosis of various cultured human cancer cells: Breast adenocarcinomas MCF-7 and MDA-MB-231, A549 lung carcinoma, Hep2 larynx epidermal carcinoma, as well as mouse cancer cells, such as Lewis lung carcinoma and HA1 hepatoma.  The efficiencies of decreasing the cell viability varied greatly for different cell lines, but cancer cells of any gynecologic malignancy have not yet been tested with the lactaptin analog. Here, we established three short-term cultures of primary endometrial tumors, one cell culture of endometrial hyperplasia cells, and a cell culture of normal endometrial tissue to reveal the sensitivity of individual endometrial cancers to the recombinant analog of lactaptin.
| > Materials and methods|| |
Patients and tissue specimens
All tissue samples (normal, hyperplasia, and tumor) were obtained with informed consent from patients at the Novosibirsk Region Oncologic Dispensary, Novosibirsk, Russia. Normal human endometrial tissue and endometrial hyperplasia were obtained from patients aged 35 and 28 years, respectively, who were undergoing diagnostic curettage. Surgical tumor specimens were obtained from three postmenopausal patients (average of 56 years) diagnosed with cancer, and had under gone complete resection of the tumors. The final diagnosis was confirmed by hematoxylin and eosin staining of paraffin blocks after the operation. None of the patients received chemo- or radiotherapy before surgery. All patients gave written informed consent. The study protocol was approved by the Institute of Molecular Biology and Biophysics SB RAMS Ethics Committee in accordance with the Declaration of Helsinki of 1975. The fresh tumor and normal tissue specimens were immediately transferred into ice-cold L15 medium (GIBCO, Invitrogen) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 250 mg/mL amphotericin B.
Tissue specimens were mechanically dissociated using a scalpel and were then transferred to a solution of 20 mg/mL collagenase I (GIBCO, Invitrogen) in L-15 medium and incubated at 37°C for 1 h on a shaking incubator (Grant Bio, Keison Products). Specimens dissociated into single cells were washed with 10 × excess of phosphate-buffered saline (PBS) and separated cells were collected by centrifugation at 300g. Cells were plated in Iscove's Modified Dulbecco's Medium (IMDM) with 10% fetal bovine serum (FBS) and, after cell adhesion, 10 μM rho kinase (ROCK) inhibitor was added to the culture medium for 1 hand the medium in plates was replaced with fresh complete IMDM medium. At the next passages, cells were cultured in complete IMDM medium supplemented with epithelial cell growth supplement (# 6622, Cell Biologics), MITO + Serum Extender (BD Bioscience - Discovery Labware), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 250 mg/mL amphotericin B and were cultivated in six-well plates at 37°C in a humidified atmosphere containing 5% CO 2 . When reaching 70-80% confluence, cells were harvested using 0.05% tripsin/EDTA (Sigma) and subcultured for further experiments.
A 2 mM stock solution of cisplatin (Sigma Aldrich, St Louis, MO) and 0.5 mM stock solution of doxorubicin (Sigma Aldrich, St Louis, MO) were prepared in sterile water and ethanol, respectively. Anastrozole and exemestane were obtained from Sigma-Aldrich and dissolved in sterile water to 2 mM concentration.
Ribonucleic acid (RNA) Extraction and Analysis
Total RNA was extracted from established primary endometrial cultures and MCF-7 cells using TRIzole reagent (Invitrogen, Carlsbed, CA) according to the manufacturer's protocols. Each sample for real-time reverse transcription polymerase chain reaction (RT-PCR) consisted of 1 μg of total RNA, 30 pmol of primers and "Master Mix RT" (BioLabMix, Novosibirsk, Russia). Real-time RT-PCR was run on a Bio-Rad iQ5 Cycler (Hercules, CA, USA) and the data were analyzed using the iQ5 system software (Bio-Rad). The relative quantification was given by the ratio between the mean value of the target gene and the mean value of the reference gene (GAPDH) for each sample. The mean threshold cycle (C t ) ratio (± standard deviation (SD)) of three independent experiments is presented. The following gene-specific primers were used for analysis: GAPDH1 5'-GAAGGTGAAGGTCGGAGT-3' and GAPDH2 5'-GAAGATGGTGATGGGATTTC-3', ERα1 5'-ATGATGAAAGGTGGGATACGA3-' and ERα2 5'-CTAGTTTGCGAGATTCTTCTTGTC-3', ERβ1 5'-GTCACAGCGACCCAGGAT-3' and ERβ2 5'-CTTACTTCTACCTCTGAGAAAAC-3', PR1 5'-TCATTCTATTCATTATGCCTTACCA-3' and PR2 5'-GAAAACCTTCCCGATGCTTCAG-3', and Cyp19-1 5'-ATGAGGGCACATCCTCAATACC-3' and Cyp19-2 5'-GTTGTAGTAGTTGCAGGCACTG-3'.
Cell proliferation/cytotoxicity assay
(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H- tetrazolium bromide) (MTT) Assay
Cell viability was determined after 72 h of incubation with drugs by MTT assay, as described previously. 
Cell proliferation and survival was monitored real-time through the iCELLigence Real Time Cell Analyser (RTCA) system (ASEA Biosciences) by measuring cell-to-electrode responses of the cells seeded in eight-well E-plates with the integrated microelectronic sensor arrays. Cells were seeded at a density of 1,500 cells per well in a total volume of 200 μL of IMDM and were monitored real-time for no less than 110 h. For the cytotoxicity assay, after the initial 24 h of proliferation, the culturing medium was replaced by fresh medium with dissolved drugs and were monitored real-time for 78-106 h.
Cell index (CI) was calculated for each E-plate well by RTCA Software 1.2 (Roche Diagnosis, France). The graphs are real-time generated outputs from the iCELLigence system.
Assessment of apoptosis
Plasma membrane phosphatidylserine exposure was investigated by flow cytometry using the BD Pharmigen Apoptosis Detection Kit (BD Biosciences). Here, 2 × 10 5 cells per well were seeded into six-well plates in complete medium and treated 24h later with RL2 or cisplatin. After 24h of treatment, cells were harvested with trypsin, washed in PBS with trypsin inhibitor (Sigma-Aldrich) and pelleted; about 1 × 10 5 cells were resuspended in incubation buffer and incubated with annexin V-fluorescein isothiocyante (FITC) and propidium iodide (PI) according to the manufacturer's protocol. Samples were analyzed on a FACSCantoII flow cytometer (Becton Dickinson) using FACSDiva Software (BD Biosciences). Intact (non-stained) cells were gated in the forward/side scatter cytometry (FSC/SSC) plot (P1) to exclude small debris.
Student's t-test was used to compare treatment effects in cell experiments. P < 0.05 were considered statistically significant.
| > Results|| |
We have established short-term endometrial primary cultures from human samples: Three endometrial cancer cells-EC1, EC2, and EC3; endometrial hyperplasia cells (HE), and normal cells (KE). After isolating the endometrial cells according to the described method, cells were grown for approximately 1 week to reach 70-80% confluence and were stained with hematoxylin and eosin solution. To sustain the viability of cancer cells in vitro we used mitogenic and growth factors recommended for epithelial cell cultures: MITO + Serum Extender and epithelial cell growth supplement. Morphological analysis of growing cells revealed their heterogeneity with the appearance of polygonal cells defined as epithelial and elongated cells defined as fibroblastoid [Figure 1]. Using the iCELLigence microelectronic biosensor system, the real-time analysis of proliferation of established cell cultures was performed. Curves of cell growth demonstrated that KE normal cells had the longest doubling time [Figure 2].
|Figure 1: Morphological analysis of growing primary endometrial cells. Cells were grown on culture slides (Falcon) and then stained with hematoxylin and eosin|
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|Figure 2: Comparison of cell proliferation. iCELLigence data showing typical cell index curves (CI) of primary endometrial cells reflected their proliferation in real-time mode. Cells were seeded at 1,500 cells per well|
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Steroid receptors and aromatase messenger RNA (mRNA) expression
To evaluate ERs, PRs, and aromatase molecular profiles in established cells, we assessed mRNA expression of ERα, ERβ, PR, and Cyp19 and scored them with the ER-positive MCF-7 human adenocarcinoma cells. The human GAPDH gene was used as an internal control and the fold-change in expression for the genes of interest was determined using the delta C t method. Normalized mRNA expression of target genes in MCF-7 cells was taken to be 100%. Real-time PCR analysis revealed that all endometrial primary cultures were ERα-positive with the same level of ERα mRNA expression, except for endometrial hyperplasia cells, which were ERα-negative [Figure 3]. All cells were ERβ- and PR-positive [Figure 3]. We found that Cyp19 mRNA expression was different for endometrial primary cultures: EC3 cells were strongly Cyp19-positive, EC1 cells were weakly Cyp-19-positive, and other cells showed no aromatase mRNA expression.
|Figure 3: Expression of ERα, ERβ, PR, and aromatase mRNA in primary cultures. Levels of transcripts were analyzed by real-time RT-PCR with specific primers and normalized to GAPDH mRNA.* and **Significantly different from MCF-7 cells. ER = Estrogen receptor, PR = progesterone receptor, mRNA = messenger ribonucleic acid, RT-PCR = reverse transcription polymerase chain reaction|
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Induction of apoptosis of endometrial cells by RL2
Endometrial cancer cells were treated with RL2, doxorubicin, and cisplatin for 106 h and cell proliferation was monitored in real time. We observed that the CI, reflecting cell numbers, started to decline after about 24 h of incubation with doxorubicin and after 35 h of incubation with cisplatin or RL2. Doxorubicin completely suppressed cell viability by 106 h of incubation, whereas cisplatin and RL2 decreased cell viability partially by that time [Figure 4].
|Figure 4: Typical curves of real-time monitoring of drug-mediated cytotoxicity. Endometrial cancer cells were seeded at 1,500 cells per well. Doxorubicin (0.3 μM), cisplatin (2 μg/mL), and recombinant analog of lactaptin RL2 (0.2 mg/mL) were added (indicated by arrow) 24 h later. Impedance at well's bottom was measured every 2 min. *Significantly different from control, P < 0.05. NS = No significant difference between groups (P > 0.05). Experiments were performed in triplicate|
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To calculate IC 50 values, MTT analysis of treated normal and cancer endometrial cells was performed. The results indicated that all cancer cells have similar IC 50 values for RL2, with the lowest reported for ER2 cancer cells. KE normal cells have the highest IC 50 values for RL2, as well as for doxorubicin and cisplatin [Table 1].
|Table 1: Evaluation of suppression of viability (IC50) of primary human endometrial cells treated with doxorubicin, cisplatin, and recombinant analog of lactaptin RL2|
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Plasma membrane phosphatidylserine exposure is one of the early signs of apoptosis.  Annexin V/PI staining and a fluorescence-activated cell sorting (FACS) analysis of RL2-treated endometrial cells was performed to estimate apoptotic cells. The Annexin V-positive cell population is composed of early apoptotic cells (annexin V + /PI− ) and secondary apoptotic or necrotic cells (Annexin V+/PI+). There was a decrease in the number of living cells after 24 h of incubation with RL2 for all endometrial cancer cells (data not shown) and the annexin V + /PI− (P2) population was more considerable for EC3 cells [Figure 5].
|Figure 5: RL2 induces apoptosis in primary endometrial cancer cells. Cells were incubated with recombinant analog of lactaptin RL2 (0.2 mg/mL) for 48 h, and then cells were assayed for apoptosis by flow cytometry using annexin V/PI staining. Typical histograms are presented. PI = Propidium iodide|
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Inhibition of cells growth by exemestane and anastrozole
The effects of exemestane and anastrozole on the proliferation of primary cultures EC3, HE, and cancer cell line MCF-7 were determined by MTT assay. Cells were treated with different doses of exemestane (1-1,000 μM) and anastrozole (2-500 μM) for 48 h. The IC 10 and IC 50 values (the drug concentration required for killing 10 or 50% of the cells, respectively) of these agents are presented in [Table 2].
|Table 2: IC50 and IC10 values of exemestane and anastrozole in MCF - 7, EC3, and HE cells|
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| > Discussion|| |
Tumor-derived cell lines play an important role in the investigation of cancer biology and genetics. Moreover, primary cell cultures prepared from cancer tissue can provide a personalized approach for the identification of effective second-line agents and to predict clinical outcomes of tumor chemotherapy.  In particular, this approach allows the efficiency of chemotherapy of heterogeneous endometrial cancer to be increased, because it is based on the biological features of the patient's tumor.
Here, we established three new human endometrial carcinoma cell lines, all derived from well-differentiated primary adenocarcinoma, one normal epithelial endometrial cell line, and cells derived from endometrial hyperplasia. It is known that mitogenic and growth factors are important for primary culture proliferation; therefore, we used commercially available medium supplements for cell culture. We have demonstrated that all cells grow well as adhesive cultures in vitro and that the proliferation rate of normal endometrial cells was noticeably lower then cells originating from neoplasm.
We have previously shown that RL2 efficiently induced apoptosis of estrogen-dependent MCF-7as well as hormone-independent MDA-MB-213 human breast adenocarcinoma cells in vitro.  Estrogens are regarded to be involved not only in the neoplastic transformation of mammary glands, but the endometrium also, and both neoplasms are considered "estrogen-dependent malignancies".  ERα plays an important role in the differentiation and proliferation of epithelial cells, whereas the role of ERβ was proposed to act as an ERα modulator, exerting an antiproliferative function in the uterus.  However, utilization of the imbalance in ERα/ERβ values between normal and cancerous tissue for an explanation of tumor progression is still being discussed. , Generally, the presence of ERα in endometrial carcinoma is associated with a less aggressive phenotype. We have compared the morphological features and mRNA expression of ERs and PRs in the established primary cell cultures. All of the investigated endometrial carcinoma cells were ER- and PR-positive and can be described as Type I estrogen-dependent carcinomas. Normal endometrial cells were also ER- and PR-positive, whereas cells of endometrial hyperplasia were ERα-negative.
The expression of aromatase, the enzyme involved in estrogen synthesis, is considered another pathophysiological factor related to the development of endometrial cancer.  Watanabe et al., demonstrated that aromatase expression, both at the protein and mRNA levels in endometrioid endometrial carcinoma are detected in stromal cells but not in carcinoma cells.  Subsequent aromatase expression in stromal cells was found to correlate with poor prognosis in women with endometrial cancer, indicating a significant role of aromatase in tumor progression.  Aromatase inhibitors show promise in the therapy of endometrial carcinoma exhibiting aromatase activity, especially in the early stage.  We revealed that only one of the primary endometrial cancer cells, EC3, expressed aromatase mRNA at a level close to that of MCF-7 cells. These findings indicate that EC3 primary cells originating from early stage carcinoma could be treated with aromatase inhibitors. To test this hypothesis we used third-generation aromatase inhibitors: The nonsteroidal anastrozole, which inhibits aromatase selectively and reversibly, and the steroidal exemestane which is an irreversible inhibitor of this enzyme. Indeed, EC3 cells were more sensitive to low concentration of aromatase inhibitors than HE cells, but less sensitive than MCF-7 cells.
At present, surgery is still the primary treatment for endometrial cancer. Other treatment options are hormonal therapy, radiotherapy, chemotherapy, and target therapy. In stage I endometrial cancer, the role of radiotherapy following hysterectomy remains controversial.  Chemotherapy with cisplatin/doxorubicin/paclitaxel, or, if tolerated, the doublet of carboplatin/paclitaxel is used more frequently as the first-line treatment of advanced endometrial cancers.  Treatment with hormones following primary surgery is thought to be beneficial in patients with endometrial cancer. Nevertheless, the decision to use any type of hormonetherapy should be individualized to reduce non-endometrial cancer-related deaths. , From the other hand, for young women who desired fertility-sparing therapy conservative treatment of well-differentiated endometrial adenocarcinoma could be alternative to hysterectomy.  Some authors showed progestin therapy to be a good choice for women having fertility desires. The reproductive outcome in cohort treated with progestin was 34.8%, but in 25.4% of women with Grade 1 carcinoma disease persistence was observed.  Thus, the development of new anticancer therapeutics with high efficiency and low toxicity is still necessary even for well-differentiated endometrial cancer.
For the study of RL2 cytotoxicity against primary endometrial cultures from individual tumors, we used doxorubicin and cisplatin as comparative drugs. The RL2 sensitivity assay verified the enhanced resistance of KE normal cells to a recombinant analog of lactaptin in comparison with cancer cells. These data supported our early findings that lactaptin predominantly induced the apoptotic death of cultured cancer cells but not normal human cells. We discovered that the IC 50 for doxorubicin was in the range of 0.16-0.29 μM for endometrial cancer cells and it was higher for nontransformed cells. Our data indicate that all established endometrial cancer cells could be considered doxorubicin-sensitive. Analysis of cisplatin IC 50 values revealed that EC1 cells were more resistant among cancer cells. We have previously demonstrated that RL2 IC 50 for estrogen-positive MCF-7 breast adenocarcinoma cells was 0.2 mg/ml. Here, computed RL2 IC 50 values were higher for all established cancer cells; close to 0.35 mg/mL. Real-time monitoring showed that only doxorubicin completely decreased viability of EC3 cells in concentration close to IC 50 value. RL2 as well as cisplatin has limited effect on viability of EC3 cells at concentration close to IC 50 value. The same limited RL2 effect was observed for apoptosis induction in EC3 cells. What's more likely, the primary cancer cell lines are not homogeneous and not all cells are sensitive to RL2. While the effectiveness of lactaptin analog alone is at time modest, further investigation of this novel agent in combination with standard therapeutics may prove to be the most fruitful approach.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]