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Year : 2020  |  Volume : 16  |  Issue : 6  |  Page : 1302-1308

Typhonium flagelliforme extract induce apoptosis in breast cancer stem cells by suppressing survivin

1 Biomedical Science Doctoral Program, Medical Faculty, Diponegoro University; Stem Cell and Cancer Research Laboratory, Medical Faculty, Sultan Agung Islamic University, Semarang, Indonesia
2 Department of Surgery, Medical Faculty, Diponegoro University, Semarang, Indonesia
3 Department of Pathobiology, Medical Faculty, Airlangga University, Surabaya, Indonesia
4 Department of Anatomical Pathology, Medical Faculty, Diponegoro University, Semarang, Indonesia

Date of Submission21-Jan-2020
Date of Decision07-Apr-2020
Date of Acceptance22-Jun-2020
Date of Web Publication18-Dec-2020

Correspondence Address:
Agung Putra
Stem Cell And Cancer Research, Medical Faculty, Sultan Agung Islamic University, Kaligawe Raya Km. 4 Semarang Central Java 50112, PO Box 1054/SM, Semarang, Central Java
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_85_20

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 > Abstract 

Context: Breast cancer stem cells (bCSCs) are a small population of cancer-initiating cells within breast cancer, characterized as CD44+ CD24–/low. bCSCs develop apoptosis resistance by expressing survivin and suppressing caspase-9 and caspase-3 expression. Typhonium flagelliforme tuber extract (TFTe) can induce apoptosis in several types of cancer cells; however, the effects of TFTe to induce the bCSCs remain unclear.
Aims: This study aimed to investigate the effects of TFTe on apoptosis induction in bCSCs through the suppression of survivin and the exhibition of caspase-9 and caspase-3.
Settings and Design: This study employed a posttest only, control group design.
Subjects and Methods: To analyze the apoptotic index, TFTe, at concentrations of 25 (Tf1d), 50.89 (Tf2d), and 100 μg/mL (Tf3d) were used to treat bCSCs for 24 h, in a humidified incubator containing 5% CO2, at 37°C. The control group was exposed to dimethyl sulfoxide. Apoptosis was measured by propidium iodide and acridine orange double-staining, and the expression levels of survivin, caspase-9, and caspase-3 were assessed by immunocytochemistry.
Statistical Analysis Used: Differences were analyzed by the independent Student's t-test, to compare two groups, and the Kruskal–Wallis test, to compare more than two groups. P < 0.05 was considered statistically significant.
Results: TFTe inhibited bCSC proliferation, with an IC50 value of 50.89 μg/mL, and significantly induced apoptosis in bCSCs (P < 0.001). TFTe also significantly decreased the expression levels of survivin in bCSCs (P < 0.001) and increased the expression levels of caspase-9 and caspase-3 (P < 0.001).
Conclusions: TFTe can induce apoptosis in bCSCs by decreasing survivin expression levels and increasing the levels of caspase-9 and caspase-3.

Keywords: Breast-cancer stem cells, survivin apoptosis, Typhonium flagelliforme

How to cite this article:
Putra A, Riwanto I, Putra ST, Wijaya I. Typhonium flagelliforme extract induce apoptosis in breast cancer stem cells by suppressing survivin. J Can Res Ther 2020;16:1302-8

How to cite this URL:
Putra A, Riwanto I, Putra ST, Wijaya I. Typhonium flagelliforme extract induce apoptosis in breast cancer stem cells by suppressing survivin. J Can Res Ther [serial online] 2020 [cited 2021 Dec 4];16:1302-8. Available from: https://www.cancerjournal.net/text.asp?2020/16/6/1302/303900

 > Introduction Top

Human breast cancer (hBC) is the most common malignancy identified in women, worldwide, with a mortality rate of 17.71 per 100,000 women among Hispanics in the United States.[1] Currently, hBC represents a major public health problem due to the emergence of apoptotic resistance among breast cancer cells in response to most currently available chemotherapeutic agents.[2] The novel mechanism of apoptosis resistance in hBC is caused by the existence of breast cancer stem cells (bCSCs), which demonstrate a robust ability to release antiapoptotic proteins, particularly survivin, which inhibit the activation of proapoptotic proteins, such as caspase-9 and caspase-3.[3] Although almost all of the artificial therapeutic agents used during cancer treatments affect bCSCs to some degree,[4] they are associated with toxic side effects, causing damage to normal cells, and have high relapse potential, which remain critical problems for currently available treatments. Therefore, the induction of apoptotic programming in cancer stem cells (CSCs) represents a promising strategy for cancer elimination, including hBC.

The small population of bCSCs in breast cancer is characterized by the cell-surface marker phenotype CD44+ CD24−/low.[5] bCSCs present stem cell properties by self-perpetuate, through self-renewal mechanisms, and generate diverse mature cell types, through differentiation processes.[2] bCSCs also display the ability to form mammospheres (spheroids), in vitro , under certain conditions.[6] Stem cell self-renewal in many stem cells, including bCSCs, can be enhanced under hypoxic conditions, due to these bCSCs can express certain genes more robustly, including those necessary for regenerative mechanisms.[7] These genes are also involved in drug resistance,[8] by presenting ATP-binding cassette transporters, activating DNA-repair and increasing anti-apoptosis proteins such as survivin, which can cause these cancer cells to become resistant to most chemotherapy treatments.[9] The ability of survivin to inhibit apoptosis was demonstrated by a previous study, which reported that survivin can suppress to activated caspases, effectively inhibiting cell death pathways.[10] Survivin also regulates the mitotic spindle checkpoint in the cancer cell cycle.[11] These findings indicated that survivin is one of the most pivotal anti-apoptosis proteins responsible for cancer cell survival, including bCSCs. Therefore, inhibiting the drug-resistance mechanisms of the bCSC may represent a critical component of successful breast cancer therapy.

The use of natural plants, which contain substances that have medicinal features but are less toxic to healthy tissues and organs, represents a potential approach to decreasing apoptotic resistance in bCSCs.[12] Typhonium flagelliforme (TF) is an herbal medicinal plant that has been widely applied during various anti-cancer studies.[13] A previous study reported that TF tuber extract (TFTe) contains unsaturated aliphatic compounds, particularly linoleic acid (LA), which has been demonstrated to induce apoptosis in various cancer cell lines.[14] The apoptosis-inducing capabilities of LA were also demonstrated by a separate study, which reported that LA-induced apoptosis by binding peroxisome proliferator-activated receptor γ (PPARγ) in CSCs. The activated LA-PPARγ complex initiates the activation of the caspase-9 and caspase-3 cascades and inhibits the survivin release, leading to apoptosis.[15] The anti-cancer in vitro effects of TF have been examined in various cancer cell lines. However, whether TF can induce apoptosis in bCSCs remains unclear. This study aimed to examine the effects of TFTe on apoptosis induction in bCSCs, by analyzing the expression levels of survivin, caspase-9, and caspase-3, as markers of apoptotic pathway activation.

 > Subjects and Methods Top

Plant materials and extraction methods

TF (Lodd.) Blume (Araceae) tubers, from Java, Indonesia, were scientifically identified by a botanist in the Faculty of Biology at Gadjah Mada University and used to produce a dichloromethane extract using the maceration method. In brief, fresh tubers were washed, cut into fine pieces, and dried in an oven (models 100–800; Memmert GmbH + Co. KG, Schwabach, Germany). The fully dried tubers were powdered and macerated with hexane for 3 days, to remove nonpolar fractions, before being treated with dichloromethane for 7 days, with occasional shaking (model KS-15; Edmund Bühler GmbH, Hechingen, Germany). Finally, the extracts were filtered and vacuum-evaporated, using a rotary evaporator (R-210; BÜCHI Labortechnik AG, Postfach, Switzerland).

Breast cancer stem cells isolation by magnetic-activated cell sorting method

bCSCs isolation was performed at the Stem Cell and Cancer Research Laboratory, Indonesia. To separate bCSCs from other subpopulation of breast cancer cells, the MACS method was used. In brief, suspensions of MCF-7 cancer cell lines were centrifuged at 300 ×g for 10 min, to obtain cell pellets. The cell pellets were resuspended in 50 μL buffer, containing 10 μL CD24 biotin, and incubated for 15 min at 2°C–8°C. The mixed cells were washed and resuspended in 80 μL buffer to produce cell suspensions. The cell suspensions were incubated with 20 μL anti-biotin microbeads and then resuspended in 500 μL buffer solution. To obtain the CD24 MCF-7 cell fraction, 500 μL buffer was added to an MS column, followed by the cell suspensions. To isolate the CD44+ cell fraction, the CD24 cell fraction was centrifuged, resuspended in 80 μL buffer, and incubated with 20 μL CD44 microbeads, for 15 min at 2°C–8°C. Cell suspensions were then applied to an MS column by flushing out to obtain the CD24/CD44+ cell fraction.

Flow cytometry to measure CD24 and CD44 expression

The expression levels of CD24 and CD44 in bCSCs were analyzed using flow cytometry. At least 105 bCSCs were centrifuged, at 500 × g, for 5 min at 4°C, to yield cell pellets. Cell pellets were resuspended in 10 μL of a monoclonal, mouse anti-human CD44–phytoerythrin antibody (BD Biosciences, San Jose, CA, USA) and a monoclonal mouse anti-human CD24–fluorescein isothiocyanate antibody (BD Biosciences, San Jose, CA, USA) and incubated for approximately 20 min, at 4°C. Flow cytometry was performed, using a Calibur flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Mammosphere formation, as a breast cancer stem cells hallmark

To induce mammosphere formation, several isolated bCSCs (CD24/CD44+) were plated in Dulbecco's modified Eagle medium-F12 medium, supplemented with 1% bovine serum albumin, 1 μM insulin, 10 ng/mL basic fibroblast growth factor, 20 ng/mL epidermal growth factor, and B-27 supplement, in a low-cell-binding dish. Mammospheres appeared as tight, round spheres floating in the medium.[16]

Cell viability assay

The toxicity profiles of TFTe were assessed, using the 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) microculture tetrazolium viability assay. bCSCs were plated in a 96-well plate, at 5 × 104 cells/well, in 20 μL medium, containing 10% heat-inactivated fetal bovine serum, and maintained in a humidified incubator, with a 5% CO2 atmosphere, at 37°C. TFTe was dissolved in 0.1% (v/v) dimethyl sulfoxide (DMSO) solution, at various concentrations (15.62–500 μg/mL). For the positive control, we used DMSO only, whereas a cell-free well was used as the negative control. After 68 h of incubation, MTT (5 mg/mL−1) was added to each well and incubated for an additional 4 h. Finally, 150 μL DMSO was added to each well to solubilize the formazan crystals. Cell viability was determined by the absorbance at 595 nm, using a microtiter plate reader (Tecan Sunrise Basic, Groedig, Austria). Percent cell viability was calculated, considering the appropriate controls, and the concentration that inhibited 50% of cellular growth (IC50 value) was determined. The cell proliferation inhibitory rate was calculated using the following formula: growth inhibition = OD control − OD treated/OD control × 100.

Apoptotic analysis of breast cancer stem cells

The apoptotic effects of TFTe treatment on bCSCs were assessed using the PI and AO double-staining technique and a fluorescence microscope (Leica Biosystems, Wetzlar, Germany). In short, 5 × 104 bCSCs/mL were seeded on amino propyltriethoxy silane (APS)-coated slides (Matsunami, Osaka, Japan) and treated with the following TFTe concentrations: 25 μg/mL (Tf1d, < IC50), 50.89 (Tf2d, IC50), and 100 μg/mL (Tf3d, > IC50). Cells were incubated in an atmosphere containing 5% CO2 at 37°C, for 24 h. To analyze apoptosis in bCSCs, 10 μL of fluorescent dyes, containing AO (10 μg/mL) and PI (10 μg/mL), was added to bCSC slides, and the cells were observed under a fluorescence microscope. More than 200 cells were examined to analyze the percentages of viable, early apoptotic, and late apoptotic or necrotic cells, as follows: Viable cells appeared homogeneously green, with intact structures; apoptotic cells contained bright green dots in the nucleus (indicating chromatin condensation), membrane blebbing, and apoptotic bodies; and necrotic or late apoptotic cells displayed orange-colored nuclei. All treatments were performed in triplicate (n = 3).

Expression of caspase-3, caspase-9, and survivin

Briefly, 5 × 104 bCSCs were seeded on APS-coated slides (Matsunami, Osaka, Japan), treated with TFTe (Tf2d = 50.89 μg/mL; IC50), incubated in a 5% CO2 atmosphere, at 37°C, for 24 h, and observed under an inverted microscope. After incubation, the cells were fixed with 4% paraformaldehyde for 10 min, washed with phosphate-buffered saline, and permeabilized with 0.1% Triton X100, for 10 min. Antigen retrieval for caspase-3, caspase-9, and survivin was performed by microwaving 10 mM citrate buffer (pH 6.0) for 20 min. Subsequently, endogenous peroxidase was inactivated by incubation with 3% H2 O 2 in methanol, for 10 min. The cells were treated with 10% normal goat serum (Nichirei, Tokyo, Japan) for 10 min and incubated with primary antibodies against caspase-3, caspase-9, and survivin (Rabbit polyclonal, 1:100; Abcam, Cambridge, UK), at 4°C overnight. Antibody binding was visualized using the secondary antibody, biotin-conjugated anti-rabbit immunoglobulin G (Nichirei, Tokyo, Japan), and peroxidase-conjugated streptavidin (Nichirei, Tokyo, Japan), followed by diaminobenzidine (DAB) solution (Metal-Enhanced DAB Substrate kit; Thermo Fisher Scientific, Waltham, MA, USA). The cells were counterstained with fresh hematoxylin (Muto Pure Chemicals Co., Ltd., Tokyo, Japan), dehydrated, and mounted. The positive expression of caspase-3, caspase-9, and survivin was marked by a brown color in the bCSC cytoplasm.

Statistical analysis

All experiments were repeated at least three times and 200 cells were examined for each plot during the immunocytochemical staining and apoptotic analyses. The results are expressed as the mean ± standard deviation. Differences were analyzed by independent Student's t-test, to compare two groups, and the Kruskal–Wallis test, to compare more than two groups. P < 0.05 was considered statistically significant.

 > Results Top

The potential effects of TFTe on apoptosis, the expression of survivin, caspase-9 and caspase-3 in bCSCs were examined in vitro , using bCSCs derived from MCF-7 cells by a MACS-based sorting method.[17] The isolated bCSCs demonstrated several CSC properties, such as anchorage-independent growth in soft agar, to form mammosphere colonies, and a CD44+/CD24 immunophenotype.

Flow cytometry analysis of CD44+/CD24 breast cancer stem cells

To confirm that all studies were performed on bCSCs, we examined the cell-surface antigen expression of CD44 and CD24 in the isolated cells, using flow cytometry. The expression of CD44 and the lack of CD24 expression are the primary surface marker characteristics of bCSCs. High-level CD44 expression has been associated with cancer progression, whereas low-level CD24 expression has been associated with nondifferentiated cells. The percentages of CD44+/CD24−/low cells were determined using dot-plots, which are illustrated in [Figure 1].
Figure 1: Flow cytometry detection demonstrates the positive expression of CD44 (83.65%) and the negative expression of CD24 (11.35%)

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Assessment of Typhonium flagelliforme tuber extracts anti-proliferative activity in breast cancer stem cells

The anti-proliferative activities of various TFTe doses were assessed by MTT assay, which measures mitochondrial dehydrogenase activity. The MTT assay is a standard colorimetric assay used to assess cellular growth. Various doses of TFTe (15.62–500 μg/mL) were used to treat bCSCs for 24 h, and the results showed that TFTe inhibited cell proliferation in a dose-dependent manner. Based on the IC50 value, TFTe exhibited anti-proliferative activity in bCSCs at 50.89 μg/mL [Figure 2].
Figure 2: Percentage growth inhibition in breast cancer stem cells after treatment with various concentrations of Typhonium flagelliforme tuber extract (15.62–500 μg/mL). The results are presented as the mean ± standard deviation for n = 3. The IC50 value was 50.89 μg/mL

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Induction of apoptosis in breast cancer stem cells

Apoptosis induction in bCSCs following TFTe administration was examined using a propidium iodide acridine orange (PI/AO) double-staining assay and immunofluorescence analysis. This method can distinguish early apoptotic cells from necrotic cells. Living cells are stained with PI, whereas dead cells are stained with AO because AO binds tightly to nucleic acids in damaged cells but cannot access the nuclei of living cells. [Figure 3]a shows the presence of viable cells in the control group (untreated group), whereas [Figure 3]b shows a proportion of cells undergoing early apoptosis.
Figure 3: Double immunofluorescence analysis of apoptosis in Breast cancer stem cells. Representative images are shown for (A) the control group, which displays homogenously green (viable) cells, and (B1 and B2) the TF2d treatment group, which displays blebbing membrane (white arrow) and chromatin condensation (blue arrow). Magnification ×200 (a) The apoptotic index values for Breast cancer stem cells after TF1d (12.47%), TF2d (22.40%), and TF3d (3.00%) treatment. The bar indicates the mean ± standard error of the mean; **P < 0.001 versus control, using the Kruskal–Wallis test (b)

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Expression levels of caspase-9, caspase-3, and survivin

The expression levels of caspase-9, caspase-3, and survivin were examined following TFTe administration and were detected as brown-colored areas in the cytoplasm of bCSCs. The overexpression of survivin in bCSCs can block the activation of caspase-9, which is an intrinsic initiator of apoptosis, and thus, inhibit bCSC apoptosis. In this study, we found the increase of caspase-9 and caspase-3 expression, as well as the decrease of survivin expression that indicated that apoptosis has been triggered, as shown in [Figure 4].
Figure 4: Immunocytochemical analysis of caspase-3, caspase-9, and survivin expression patterns in Breast cancer stem cells. Representative images are shown for the control group (A1, B1, and C1) and the TF2d treatment group (A2, B2, and C2). Closed arrows indicate positive expression in cells (brown color in the cytoplasm). Magnification × 200 (a). The percentage of caspase-3-positive cells (29.80 ± 5.85%), caspase-9-positive cells (21.87 ± 5.97%), and survivin-positive cells (2.60 ± 0.57%) after Tf2d treatment; **P < 0.001 (b)

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 > Discussion Top

hBC is the second-leading cause of cancer-related deaths in women, worldwide,[18] and bCSCs are thought to play a pivotal role.[19] The ability of bCSCs to resist apoptosis induction in response to most cancer treatments suggests that investigating these cells and understanding the resistance mechanism is crucial.[2],[20] Increased survivin expression appears to be one of the primary means used by bCSCs to block the apoptosis pathway,[21] which makes bCSCs difficult to eliminate using most conventional treatments. In this study, we aimed to interfere with bCSC viability through the administration of various doses of TFTe, to induce apoptosis. We examined survivin, caspase-9, and caspase-3 expression to determine whether bCSC death following TFTe administration was caused by apoptosis or necrosis. To isolate bCSCs from the breast cancer cell line MCF-7, we used the MACS method to identify CD44+ and CD24 cells, as described by a previous study.[16]

In this study, we found that survivin was highly expressed in all control groups, which indicated that untreated bCSCs can robustly inhibit apoptosis induction. These findings agreed with those reported by a previous study, which found that increased survivin expression inhibited activated caspase-9, which is a protein involved in apoptosis initiation.[22] Therefore, the emergence of high apoptotic resistance in bCSCs may be due to the high level of survivin expression. However, we found that TFTe treatment could decrease survivin expression in all treatment groups, indicating that TFTe treatment can induce apoptosis in bCSCs. Therefore, to analyze the role played by TFTe in apoptosis induction, we determined the IC50 dose, which is the dose necessary to inhibit cell proliferation by 50%. We found that the IC50 value for TFTe in bCSCs was 50.89 μg/mL [Figure 2], based on the MTT assay results. The MTT is a standard colorimetric assay that uses changes in color to analyze the cellular growth. The American National Cancer Institute guidelines limit the activity of crude extracts at IC50 values for proliferation <100 μg/mL, after an exposure time of 24 h.[23] Therefore, we further analyzed whether bCSCs proliferation was inhibited by apoptosis or necrosis.

We found that the growth inhibition caused by TFTe treatment was due to the induction of apoptosis. Our findings showed the most of the bCSC membranes remained intact during the treatment process, which indicated that the TFTe-treated bCSCs were undergoing early apoptosis. A previous study reported that intact cell membranes following treatments may indicate that cells are in the early apoptosis stage, instead of necrosis.[24] To confirm that bCSC apoptosis occurred, we examined the morphological changes in bCSCs using AO/PI double-staining.[25] We found membrane blebbing, nuclear condensation, and apoptotic bodies in bCSCs treated with TFTe [Figure 3]a, B1 and B2]. These findings indicated that TFTe can induce apoptosis in bCSCs. However, to confirm our findings, we also assessed the expression levels of survivin, caspase-9, and caspase-3, as these represent the primary proteins associated with the apoptosis pathway.

Survivin is a well-known member of a family of apoptosis inhibitors that are primarily expressed in CSCs, including bCSCs, and is robustly correlated with apoptotic resistance.[26] We found decreased survivin expression in all treatment groups, associated with increased caspase-9 and caspase-3 expression. These results indicated that the TFTe can suppress survivin expression in bCSCs, resulting in increased caspase-9 and caspase-3 levels [Figure 4]a and [Figure 4]b, which, in turn, increases apoptosis. These findings agree with the results reported by other studies, which found that decreased survivin levels can induce increased caspase-9 and caspase-3 levels.[27],[28],[29]

Together, these findings suggested that TFTe can induce apoptosis in bCSCs. Several studies have reported that TFTe contains LA, which can induce apoptosis in most cancer cells.[30],[31] A previous study reported that TFTe contained high levels of unsaturated aliphatic compounds, particularly LA and conjugated LA,[32] which may induce bCSC apoptosis by binding to PPAR-γ, a nuclear receptor expressed in bCSCs,[33] and retinoid X receptor, forming a complex protein of PPAR response element (PPRE) in the proline oxidase (POX) promoter area.[34] The POX promoter may then induce the expression of POX that serve as inducing reactive oxygen species (ROS) formation and leading to apoptosis.[35] However, this complex may also suppress nuclear factor kappa B (NF-κB) signaling,[36] resulting in reduced antioxidant protein levels and the suppression of survivin. Decreased survivin levels can induce increased caspase-9 and caspase-3 levels, leading to apoptosis [Figure 5]. Therefore, the accumulation of ROS, combined with decreased survivin levels, may be involved in the observed increase in bCSC apoptosis.[37]
Figure 5: Typhonium flagelliforme tuber extract contained the high levels of linoleic acid and conjugated linoleic acid may bind to peroxisome proliferator-activated receptor γ of Breast cancer stem cells and retinoid X receptor to form a complex protein at peroxisome proliferator-activated receptor response element in the proline oxidase promoter area. These may induce both the reactive oxygen species formation for apoptosis induction enhancement and suppress nuclear factor kappa B signaling for the suppression of survivin. Decreased survivin levels can induce increased caspase-9 and caspase-3 levels, leading to apoptosis

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The present study has some limitations. We did not analyze the binding between LA and PPAR-γ following TFTe treatment; therefore, which genes were activated during apoptosis induction remain unknown. We also did not analyze whether the NF-κB pathway was involved in the decreased survivin expression; thus, the mechanism underlying decreased survivin expression remains unclear.

 > Conclusion Top

Based on our study, we conclude that TFTe as a traditional medicine may enhance apoptosis in BCSCs by decreasing survivin expression as anti-apoptotic protein and increasing the expresion of caspase-9 And caspase-3 as pro-apoptotic protein. These findings also suggest that the potential anticancer activities of TFTe should be further developed for cancer treatment in the future.

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Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]


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