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Abstract

The chaperone-binding drug, 17-allylamino-17-demethoxygeldanamycin, has recently come into clinical use. It is a derivative of geldanamycin, an ansamycin benzoquinone antibiotic with anti-carcinogenic effect. Understanding the effect of this drug on the cancer cells and their niche is important for treatment. We applied 17-allylamino-17-demethoxygeldanamycin to colon cancer cell line (Colo 205) on matrix molecules to investigate the relationship of apoptosis with terminal deoxynucleotidyl transferase dUTP nick end labeling immunocytochemistry and related gene expression. We used laminin and collagen I for matrix molecules and vascular endothelial growth factor for angiogenic structure. We also examined apoptosis-related signaling pathway including mitochondrial proteins, cytochrome c, Bcl-2, caspase-9, Apaf-1 expression using real-time polymerase chain reaction. There was clear effect of 17-allylamino-17-demethoxygeldanamycin that killed more cells on tissue culture plastic compared to matrix molecules. The IC₅₀ value was 0.58 µg/mL for tissue culture plastic compared with 0.64 µg/mL for laminin and 0.75 µg/mL for collagen I. The analyses showed that more cells on matrix molecules underwent apoptosis compared to that on tissue culture plastic. Apoptosis-related gene expression was similar in which Bcl-2 expression decreased and proapoptotic gene expression of the cells on matrix molecules increased compared to that on tissue culture plastic. However, the application of 17-allylamino-17-demethoxygeldanamycin was more effective for the cells on collagen I compared to the cells on laminin. There was also a decrease in angiogenesis as shown by the vascular endothelial growth factor staining. This was more pronounced by coating of the tissue culture plastic with matrix molecules. Our results supported the anti-cancer effect of 17-allylamino-17-demethoxygeldanamycin, and this effect depended on matrix molecules. This effect occurs through apoptosis, and related genes were also altered. All these genes may serve for novel target under the effect of matrix substrate. However, correct interpretation of the results requires further studies.

Keywords

17-allylamino-17-demethoxygeldanamycin Colo 205 matrix oxidative stress apoptosis

Introduction

Colon carcinoma is the third most common type of cancer, and surgery is not enough to prevent metastasis.¹ Chaperones are essential for the function of oncogenic proteins which are the targets of carcinogenesis. Geldanamycin (GA) is one of the earliest inhibitors of chaperones.² 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a semisynthetic derivative of GA and a natural inhibitor of heat shock protein 90 (HSP90). Tumor cells are especially sensitive to 17-AAG.³ The niche is a suitable cellular and stromal environment for the oncogenic development. Laminin is a matrix protein that is important for the metastasis while collagen I is also matrix molecule which regulates migration during invasion. Another important factor besides niche is oxidative stress–induced apoptosis.⁴ Oxidative damage has been attributed to the regulation of apoptotic genes.⁵ These mechanisms play key roles for the pathogenesis of proliferation and cell death.^6,7

Nitric oxide synthase (NOS) is responsible for the conversion of l-arginine to l-citruline to produce nitric oxide (NO). There are three isoforms of NOS, namely, endothelial NOS (eNOS; NOS1), inducible NOS (iNOS; NOS2), and neural NOS (nNOS; NOS3). Reactive oxygen species (ROS) are very important to produce necrotic or apoptotic cell death in many types of cancer.⁸ Inhibition of apoptosis has been shown to inhibit nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathway.⁹ These reactive molecules also contribute to oxidative damage of membrane lipids and damage to DNA which causes apoptosis by Bcl-2 and caspase activation.¹⁰

Apoptosis encompasses many processes which normally occurs during development but is also useful to eliminate carcinogenic cells. Apoptotic cell death is an important mechanism for anti-cancer drugs. Two different mechanisms exist: one from death receptors present on the cell membrane and the other from the mitochondria. The release of cytochrome c from mitochondria to cytosol is activated during mitochondria-mediated apoptosis. Following the cytochrome c release to cytosol, cytosolic apoptotic protease activating factor 1 (Apaf-1) and procaspase-9 assemble to form apoptosome.^11,12 Procaspase-9 is converted into caspase-9 in this complex and caspase-9 activates caspase-3 and caspase-7, which cause cell death.¹³ One of the most important mechanisms of apoptosis modulators is Bcl-2 gene. Bcl-2 is known as the first gene that protects cells from apoptosis.¹⁴ It exerts this anti-apoptotic effect by preventing cytochrome c release and the activation of effector protease.¹⁵ Reduction in the levels of Bcl-2 cells leads to apoptosis, whereas its increase prevents cells from dying.¹⁴ It has been shown that many anti-cancer agents induced apoptosis, and this was mediated by caspase-3 and caspase-9, which upregulate Bax proteins, resulting in an increase in release of cytochrome c to cytosol.¹⁶ Signaling proteins which have an important task in the initiation and progression of apoptosis are called chaperones. HSPs function in the regulation of cellular homeostasis and cell survival as molecular chaperones.¹⁷ HSP90 plays a role in regulating apoptosis by interacting with different proteins at critical control points and prevents apoptosis due to its cell protecting property.¹⁸ The inhibition of HSP90 by various agents causes the release of cytochrome c to cytosol which plays an important role in the initiation of apoptosis by causing mitochondrial membrane depolarization.¹⁹ 17-AAG is a derivative of geldanamycin. Many works done in colon cancer cell line showed that 17-AAG induced apoptosis in these cell lines by blocking the function of chaperone HSP90.^20–23

Tumors produce specific extracellular matrix (ECM), which provide signaling information for survival, invasion, and metastasis. Laminin and collagen are special members of ECM which are highly expressed during carcinogenesis. Laminins are structural components of basement membranes. In addition, they are key ECM regulators of cell adhesion, migration, differentiation, and proliferation.²⁴ Type I collagen settles in the extracellular space and gives hardness to the tissue and creates a biomechanical surface for macromolecules to adhere. Type I collagen is a favorable substrate for cell adhesion and growth and is remodelable by many tissue cells; these characteristics make it an attractive material for the study of dynamic cellular processes.^25,26

17-AAG, as a natural product, may be important to help complementary treatment of colon cancer. Possible mechanisms for its action are oxidative stress and apoptosis which get signals from laminin and collagen found in their niche. Therefore, we aimed to investigate the effect of 17-AAG on the cells on matrix molecules and their relation to stress and apoptosis which play important roles in colorectal cancer and its resistance to treatment.

Materials and methods

Geldanamycin application

A stock solution of 17-AAG (10 mM) was prepared by dissolving in dimethyl sulfoxide (DMSO) solution. Then this stock solution was used to prepare working solution with concentrations of 1, 3, 10, 100, and 500 µM doses to treat Colo 205 colon cancer cell line.

Cell culture

Adherent Colo 205 cell line was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) F-12 supplemented with 10% fetal calf serum (FCS), 1% l-glutamine and 1% penicillin–streptomycin containing cultural medium. Colo 205 cell line was bought from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in a humidified atmosphere at 37°C in 5% CO₂. The morphology of the cells was examined every second day using a phase-contrast inverted microscope (CK40-F200; Olympus, Tokyo, Japan) and photographed. When the cells were confluent, they were routinely sub-cultured using 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) solution.²⁷ All chemicals were from Sigma.

Matrix molecule coating

In total, 24 six-well plates were coated overnight at 4°C with 100 μL of collagen I diluted in 900 μL of 2% (v/v) acetic acid (1:10 dilution) and 100 μL of laminin diluted in 900 μL of 1 mg/mL in phosphate-buffered saline (PBS), pH 7.4 (1:10 dilution), at a concentration of 4 μg per well. Both laminin and collagen I were coated at 6.25 μg/cm².²⁸

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

The mitochondrial functions of these cells and the density of living cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) which is one of the colorimetric test methods. This test measures the conversion of MTT dye, which is yellow in color and solubilizes in water to purple formazan products via the electron current in active mitochondria. MTT stock solution (50 g MTT + 10 mL PBS, 5000 mg/mL) can be stored in the refrigerator. All PBS in solutions was adjusted to pH 7.4. Cells were seeded onto 96-well plates at a density of 10⁴ cells/well with 200 μL of medium for 24 h. The culture medium was removed from the culture wells, then 200 µL/well of MTT solution (1 mL of stock solution of MTT + 9 mL of growth medium) was added and the plate was incubated for 3 h under culture conditions. MTT solution was removed, 200 µL of DMSO was added to each well, and spectrophotometric measurements were performed using a microplate reader at 570 nm with a 690 nm reference filter.²⁹

Immunohistochemistry

Cells were fixed in 4% paraformaldehyde solution and washed with PBS (pH 7.4) three times for 5 min after fixation. Then, the cells were incubated with 0.5% trypsin solution for 5 min, followed by washing as described above, and 3% hydrogen peroxide (H₂O₂) was added and allowed to stand for 30 min. The cells were washed and incubated in blocking serum for 1 h, followed by incubating with serum anti-VEGF (sc-7269; Santa Cruz Biotechnology, Dallas, Texas), anti-eNOS (RB9279; Lab Vision, Fremont, CA), and anti-iNOS (RB9242; Lab Vision) antibodies for 18 h. After washing, biotinlayted anti-mouse/anti-human horseradish peroxidase–conjugated streptavidin was added and incubated for 30 min. Then, the secondary antibody was washed with PBS three times for 5 min. The immunoreactivities were determined by staining with diaminobenzidine (DAB; 00-2020; Zymed Laboratories, San Francisco, CA) for 5 min. Negative controls were placed in PBS instead of primary antibody. Samples were closed by mounting solution water (00-8030; Histomount mounting solution; Thermo Fisher Scientific, San Francisco, CA) after washing with distilled water.³⁰ The staining intensity of slides processed using the immunohistochemical protocol was graded semiquantitatively, and the H-score was calculated using the equation H-score = Pi(I + 1), where i = the intensity of staining, with a value of 1, 2, or 3 (weak, moderate, or strong, respectively), and Pi is the percentage of stained cells for each intensity, varying from 0% to 100%.

Terminal deoxynucleotidyl transferase dUTP nick end labeling assay

Dead End Colorimetric TUNEL system kit (G7130; Promega, Madison, WI) was used for this technique. After the cells had been incubated with 4% paraformaldehyde for 10 min, they were washed with buffer solution for three times for 5 min each. After being performed and washed with 4% paraformaldehyde, the cells that were washed with buffer solution for 5 min were incubated for 1 h with TdT-enzyme solution at 37°C. The cells were treated with 3% H₂O₂ (TA-015-HP; Lab Vision) for 5 min, then they were added with 22% NaCl and 11% sodium citrate for 10 min and washed with buffer solution for 10 min at room temperature. The cells were then incubated with anti-streptavidin-peroxidase enzyme for 30 min and were dyed with DAB. The cells were washed three times for 5 min in distilled water. They were examined under a microscope after being mounted with Histomount mounting solution.³⁰ Staining was examined independently by two histologists who had no information about the origin of the samples. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells were counted in each visual field and reported as a percentage of total cells.

RNA isolation protocol

In this study, a control group and 17-AAG-administered group were used. TriPure isolation reagent was added to the cells in the flask and the scraped cells were added to the tubes. Then they were centrifuged at 3000 r/min for 30 s. The cells were taken to the special eppendorf tubes with beads at the bottom. They were rotated at MagNA Lyser homogenizer at 3000 r/min for 30 s. The tubes were removed from the homogenizer and taken to the cooling block and allowed to stay at room temperature for 5 min. Then 200 µL of chloroform was added to the tubes and incubated for 5 min, followed by centrifugation at 12,000 r/min for 20 min at 4°C. This process resulted in the density-gradient separation of DNA, RNA, and protein in three phases: 1st phase (aqueous phase) includes RNA and is colorless; 2nd phase contains DNA and is white; and the 3rd phase (organic phase) includes proteins and is red.

For RNA isolation, it was put into 500 µL colorless phase 1 tubes. A volume of 500 µL of isopropanol was added to it. They were incubated for 10 min at room temperature and centrifuged for 10 min at 12,000 r/min at 4°C. A volume of 1 mL of 75% of ethanol was added to the precipitated form and centrifuged at 4°C for 5 min at 12,000 r/min. At the end of the centrifugation, the supernatant was discarded and the ethanol was allowed to evaporate at 57°C. Pipetage was done by adding 50–100 µL of RNAse-free water to the remaining precipitate, and thus the precipitate was dissolved.

Complementary DNA synthesis

After addition of RNAse-free water, the absorbance of the cells was measured. For each sample, a mixture of total 11.4 µL was prepared from 9.4 µL of RNA + H₂O and 2 µL of random hexamer primer. This mixture was taken into smaller tubes and pipetage was performed. Afterward, the tubes were placed in thermal cycler and incubated at 65°C for 10 min.

Master mix was prepared in the meantime. For each sample, 4 µL of the reaction buffer, 2 µL of deoxynucleotide triphosphate (dNTP), 1 µL of dithiothreitol (DTT), 1.1 µL of enzyme, and 0.5 µL of RNAse inhibitor were prepared for a total of 8.6 µL of master mix. Then, this master mix (8.6 µL) was added to the samples (11.4 µL) taken from thermal cycler, and the pipetage was performed. Final volume of complementary DNA (cDNA) samples was made up to 20 µL. Then, the tubes were placed in a thermal cycler and run at 55°C and 85°C for 30 and 5 min, respectively.

Real-time polymerase chain reaction

cDNA sample was prepared so that the final volume of the reaction mixture was 10 µL. For each sample, 3.5 mL of dH₂O, 0.5 mL of the primer probe mix, 5 mL of enzyme were prepared for 9 mL of final mixture. To this mixture, 1 µL of cDNA sample was added and pipetage was performed. The reaction mixture (10 μL) was distributed to each well of 96-well plate, and the plates were read in polymerase chain reaction (PCR). Afterward, 1 h reading activity took place in PCR. Clustal Waling and Oligo7 software were used.

Statistical analyses

The statistical analyses were performed using analysis of variance (ANOVA) method with GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA). Statistical significance (p < 0.05) was indicated by Tukey’s multiple comparisons test

Results

Colon cancer cells adhered strongly to each other on the surface of tissue culture plastic (TCP) dish and the behavior of the cells was similar on the surface coated with laminin (Figure 1). Few cells were epitheloid shaped under the effect of laminin. The proliferation of the cells was similar but faster. The toxic effect of 17-AAG was dose-dependent between 1 to 10 µM in confluent cultures with the half maximal inhibitory concentration (IC₅₀) of 8.06 µM for TCP and 21.6 µM for laminin (Figure 2).

Figure 1.

Images from inverted phase-contrast microscopy of Colo 205 cancer cells on the laminin- and collagen I–coated TCP where 17-AAG application killed almost all the cells at high concentration. Altered cell behavior was separation from each other with less cell-to-cell adhesion. Matrix molecule–coated TCP made cells more resistant to anti-cancer effect of AAG (magnification: 20×).

Figure 2.

MTT results of the AAG effect for Colo 205 cancer cells on the laminin- and collagen I–coated TCP where 17-AAG applications killed the cells in dose-dependent manner. IC₅₀ was 8.06 µM for TCP, 21.6 µM for laminin, and 26.8 µM for collagen I.

Colon cancer cells showed less adhesion to the collagen I–coated culture dishes (Figure 1) compared to uncoated dishes and they produced fewer colonies with less proliferation in cultures. Few cells showed bipolar morphology and less cell-to-cell adhesion. The IC₅₀ dose of 17-AAG on the cells of collagen I–coated surface was 26.8 µM (Figure 2).

Colo 205 cells on the matrix substrates died of necrosis but also underwent apoptosis at the dose of IC₅₀ (Figure 3). Apoptosis index of cancer cells at IC₅₀ was 14.66% ± 4.88% for laminin substrate and 52.36% ± 18.65% for laminin substrate plus 17-AAG, whereas it was 18.64% ± 8.24% for collagen I substrate and 42.76% ± 16.34% for collagen I substrate plus 17-AAG.

Figure 3.

Apoptosis labeling of Colo 205 cancer cells on the laminin- and collagen I–coated TCP. There was an increase of apoptosis for the cells on matrix molecule–coated TCP after the application of 17-AAG compared to that of cells on TCP only.

Cells in culture were stained for vascular endothelial growth factor (VEGF), and the staining intensity and localization were increased due to the substrate effect of laminin and collagen I (Figure 4). The H-score was 425.33 ± 48.96 for TCP substrate and 178.23 ± 32.44 for TCP substrate plus 17-AAG, whereas 325.65 ± 45.56 for laminin substrate and 125.65 ± 25.56 for laminin substrate plus 17-AAG application, and 388.75 ± 75.77 for collagen I substrate and 95.17 ± 22.77 for collagen I substrate plus 17-AAG.

Figure 4.

VEGF-positive cells of Colo 205 cancer cells on the laminin- and collagen I–coated TCP were increased. There was a decrease in VEGF for the cells on matrix molecule–coated TCP after 17-AAG application compared to that of cells on TCP only.

Application of 17-AAG to the colon cancer cells on the laminin and collagen I substrate altered apoptosis-related genes. Gene expression results of Bcl-2 levels were also significantly decreased (p < 0.05) under the effect of matrix molecules. However, cytochrome c, caspase-9, Apaf-1 levels were also significantly increased (p < 0.05) for the cells on laminin and collagen I substrate. Moreover, application of 17-AAG caused more increase for colon cancer cells on collagen I substrate compared to that of cells on laminin substrate (Figure 5).

Figure 5.

RT-PCR results of the apoptosis-related genes for the AAG effect on the Colo 205 cancer cells with the laminin- and collagen I–coated TCP where AAG application produced more apoptosis-related genes.

Discussion

Matrix molecules such as laminin and collagen I were used as a carpet on the TCP for the niche environment of colon cancer cell line Colo 205 to understand the effect of application of 17-AAG, which is a natural geldanamycin derivative. Its proliferative and apoptotic effects on the cancer cells in culture were investigated by inverted phase-contrast microscope. The effect of 17-AAG on the angiogenesis and apoptosis of cells was examined by immunocytochemistry. Apoptosis-related genes were also checked by real-time PCR (RT-PCR). Application of 17-AAG caused proliferation inhibition and angiogenesis along with cell death. Matrix molecules gave resistance to the cells under the effect of 17-AAG against cell death via apoptosis. Angiogenesis was also less decreased for the cells on matrix molecules after 17-AAG application.

Angiogenesis is important for the tumor growth and metastasis. Tumor cells can use signals and messengers to produce more blood cells. Tube formation assay has been used to show angiogenesis for the inhibitors or activators. Therefore, the target for the angiogenesis is the best way to stop growth, migration, and proliferation of cancer cells. Thus, 17-AAG is a candidate for this target and its effect depends on matrix molecules.³¹ We showed that this natural product produced less VEGF-positive cells on the laminin and collagen I substrate. This is important because there was less proliferation and more apoptosis after 17-AAG treatment. This behavior of the colon cancer cells could be attributed to production of fewer vessels for tumor.

Colon cancer is third commonly seen cancer worldwide.¹¹ Apoptosis is a controlled and programmed cell death through cell-associated and signal-regulated mechanism.³² Induction of cell cycle arrest and apoptosis is a potential strategy for cancer treatment. HSP expression is essential for cancer cell survival, and neutralizing HSP with 17-AAG is also a strategy to kill these cells. Matrix molecules are stromal tumor cells, which produce tumor microenvironment by the secretion of cytokine growth factors and matrix remodeling enzymes. This environment is very important for the cancer invasion and metastasis.

17-AAG connects to HSP90 and prevents its protective feature, which causes apoptosis in Colo 205,²³ similar to our results. It has been shown that increased mitochondria and death receptor–mediated apoptosis prevented the proliferation of Colo 205 cells.³³ It has also been shown that 17-AAG inhibits chaperone function of HSP90 by blocking the signal transmission of colon cancer cell lines (HCT116, HCT15, HT29), similar to Colo 205.^21,22 Bcl-2 plays a significant role in the occurrence of apoptosis. The reduction of the Bcl-2 has been reported to inhibit the mitochondrial release of cytochrome c, Bcl-2, caspase activation, and the prevention of anti-apoptotic effect including cell death.^2,34 In our study, Bcl-2 expression by the cancer cells in culture was decreased statistically after 17-AAG treatments. There was a decrease in the Bcl-2 value of the cells in other cancer types treated with 17-AAG and this was related to the inhibition of tumor growth. This was also true for 17-AAG-treated breast cancer cell line such that oncogenic proteins were decreased and cytotoxicity was increased due to cell cycle arrest and apoptosis.^3,35 The application of an antagonist to Bcl-2 and 17-AAG together caused anti-tumor activity in cancer cells, which were resistant to apoptosis due to high levels of Bcl-2 in HL-60 cell line.³⁶ Laminin and collagen I are important niche molecules, which affect membrane receptor and cytoskeleton of the cells. These molecules are secreted by the cells themselves but when they are used as carpet the effect can be much more. These relation of the cancer cells may provide immunity to anti-cancer agents such as 17-AAG.³⁶ This is important not only for inhibition of the proliferation but also for cell death. Apoptosis is regulated by mitochondria and Bcl-2, which control intrinsic pathways. The effect of 17-AAG was less prominent on the cells on matrix molecules. These alterations were confirmed by apoptotic genes. There was also an increase in the Bcl-2, overexpression of which increases cell survival.

Matrix molecules play important role in cell adhesion, which may show with spheroid embedded in matrix with three dimensional (3D) gel systems. HCT116 spheroids embedded into 3D collagen I gels show outbound movement or dissemination of single or small group of cells under the effect of lysyl–transfer RNA (tRNA) synthetase (KRS). In these gels, there were increases in E-cadherin, β-catenin, Zonula occludens-1, and mesenchymal markers such as 1α Vimentin, N-cadherin, and fibronectin. Colon cancer cells in a matrix gel such as collagen I affect migration and invasion of the cells which might be related to epithelial–mesenchymal transition depending on cadherin expression. Therefore, cell–matrix interactions may affect cell-to-cell adhesion during carcinogenesis. This might be the explanation for molecules like KRS and extracellular signal–regulated protein kinases 1 and 2 (ERK1/2) signaling to matrix for prometastatic roles. It has been suggested that matrix molecules might cause dissemination of the cells.⁴ Loss of attachment which involves disassembly of matrix adhesion and reorganization during invasion is important for cancer treatment. Integrins, laminin, and collagen are altered during invasion. Tissue microarray analyses showed that there is a relation between laminin and collagen and the increase in these proteins in vivo is correlated with tumor metastasis.⁵ In our study, colon cancer cells on matrix molecules showed more defenses to 17-AAG treatments.

Apoptotic cell death of the cells that were inhibited by HSP90 occurs as a result of the release of cytochrome c to cytosol by mitochondrial way.³⁷ In our study, it has been shown that the expression of cytochrome c increased significantly when colon cancer cell line was treated with 17-AAG. Similar to our study, Hsu et al.³⁸ also found a reduction in the expression of Bcl-2 and an increase in the expression of cytochrome c and caspase-9 in Colo 205 cell line and argued that the cell growth was inhibited in these cells by inducing mitochondria-mediated apoptosis. In HL-60 cell line, apoptosis occurred when 17-AAG increased the mitochondrial release and accumulation of cytochrome c in the cytosol by the activation of caspase-3 and caspase-9.³⁶ Matrix molecules have negative prognostic value for colon cancer, which may be relevant to tumor progression. The effect of matrix molecules could be related to adhesion of the cells to the matrix not allowing the cells to proliferate. An alternative would be to produce a proper microenvironment with the cells such as endothelial, fibroblast, and immune cells. This niche containing these cells creates and organizes matrix networks for angiogenesis and tumorogenesis. These matrix molecules may help the cells to disseminate and therefore enhance tumor metastasis after anti-cancer treatment. These molecules decreased cell death and proliferation after 17-AAG treatment showing which matrix molecules should be a target for the anti-cancer drug.³⁹

It has been shown that apoptotic signal for the cells of Colo 205 start with caspase-8. These trigger proapoptotic molecules such as Bcl-2, Bax, and cytochrome c. This is usually due to endoplasmic reticulum stress as a result of oxidative stress.⁸ It also has been shown that oxidative stress caused not only apoptosis but also autophagy which reduced the dysfunctional cellular compartments, another target for the cancer therapy.⁴ Production of free radicals triggers the loss of matrix metalloproteinase, which causes an irreversible apoptosis.¹² Another effect of oxidative stress is the hypoxia-related cell death. Hypoxia is important for the colon cancer cells which produce hypoxia-inducing factor-1α (HIF-1α). This factor increases the VEGF and tumor vessel formation.¹⁰ The balance between cell proliferation and apoptosis is important for carcinogenesis, and apoptosis is the target for the drugs. Therefore, 17-AAG is important for this target which matrix molecules another determinant of this effect of geldanamycin. The relation between oxidative stress and apoptosis has been shown in vivo as well.^7,9 It has been reported that Apaf-1 and caspase-9 were required for the apoptosis where cytochrome c was induced by 17-AAG.^40,41 Caspases are the central components of apoptotic program.⁴² The expression of caspase-9 by cells of colon cancer cell line was increased significantly after 17-AAG. A study on bladder cancer cells reported that 17-AAG-induced cell death increased the caspase-9 and caspase-3.⁴³

In conclusion, in this study, it has been shown that the application of 17-AAG on the colon cancer line inhibits the cell proliferation, significantly increases the expression of apoptotic proteins such as cytochrome c, Apaf-1, and caspase-9, and significantly reduces the expression of anti-apoptotic protein Bcl-2. Therefore, 17-AAG appears to be a reliable agent which can contribute to the treatment of colon cancer. 17-AAG is a drug that has entered phase 1 and phase 2 studies; we thought that the use of this drug can provide a hope for colon cancer treatment.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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In vitro investigation of the effect of matrix molecules on the behavior of colon cancer cells under the effect of geldanamycin derivative

Abstract

Keywords

Introduction

Materials and methods

Geldanamycin application

Cell culture

Matrix molecule coating

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Immunohistochemistry

Terminal deoxynucleotidyl transferase dUTP nick end labeling assay

RNA isolation protocol

Complementary DNA synthesis

Real-time polymerase chain reaction

Statistical analyses

Results

Discussion

Footnotes

Declaration of conflicting interests

Funding

References