Inhibit cancer cell proliferation -
Mice were transferred to the cage when completely recovered. The water was replaced every 2 days. To evaluate tumor volume, the greatest longitudinal length and the width of tumor were measured using caliper. Mice were monitored for behavior, bodyweight, and tumor size using a vernier caliper every 2 days up to 9-week post-xenograft.
If mice demonstrated signs of pain, analgesia would be applied. However, pain and distress were not detected in any mice throughout the experiment. None of the mice died before meeting criteria for euthanasia. The confirmation of euthanasia was performed by inducing pneumothorax and removing vital organs.
The tumor cells from the primary implantation site were harvested for total protein determination by western blot analysis. The COL17A1 mRNA expression in primary breast cancer and patient clinical data from METABRIC study [ 18 ] were retrieved from cBioPortal [ 45 ].
The level of COL17A1 expression was categorized as low and high expression comparing to median COL17A1 expression for subsequent analysis of tumor-free period and overall survival.
Patients with unknown data of COL17A1 expression, time to tumor-free and overall survival were excluded. Cox regression model was used for univariate and multivariate analyses of the following variables: COL17A1 expression level, p53 status, patient age, tumor subtypes by 3-gene classifier, and pathological stage 0—IV.
All data were reported as mean ± standard deviation. Statistical analysis was done by GraphPad Prism 5. Median overall survival data were demonstrated as a Kaplan-Meier survival curve and the difference was compared using the logrank Mantel-Cox test. The authors would like to thank the Pharmaceutical Research Instrument Center of the Faculty of Pharmaceutical Sciences at Chulalongkorn University.
We thank Professor Pithi Chanvorachote, Assoc. Varisa Pongrakhananon and Asst. Supannikar Tawinwung for providing some laboratory materials and valuable advice. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Article Authors Metrics Comments Media Coverage Reader Comments Figures.
Abstract Collagen XVII COL17 , a cell-matrix adhesion protein, has been found to be suppressed in breast cancer. Introduction Breast cancer is the most common cancer in women and one of the leading causes of cancer death [ 1 ]. Download: PPT.
Fig 1. Fig 2. COL17 inhibits the cell proliferation, clonogenicity and growth in 2D culture. Fig 4. COL17 reduces the spheroid size and proliferation in 3D culture. COL17 delays breast cancer progression and prolongs survival in mice To evaluate the effects of COL17 on the overall survival of mice in addition to the progression of primary tumor, an MDA-MB cells were used for a xenograft.
Fig 6. COL17 delays breast cancer progression and prolongs survival in mice. High COL17A1 level is associated with low proliferation and extended tumor progression in patients The associations of COL17A1 expression and tumor progression were explored in breast cancer patients using data from the METABRIC study [ 18 ].
Fig 7. High COL17A1 level is associated with low proliferation and extended tumor progression in patients. Table 1. Univariate and multivariate Cox regression model using METABRIC database. Discussion COL17 has been reported to be as an adhesion molecule and receptor that mediates signaling pathways in skin epithelia [ 9 , 19 ], but its function in other tissues has rarely been studied.
Fig 8. Materials and methods Cell lines and treatments The original cell lines, MCF7 ATCC HTB TM and MDA-MB ATCC HTB TM , were purchased from the American Type Culture Collection. Antibodies For western blotting, anti-collagen XVII ab and HRP-conjugated secondary antibody ab were purchased from Abcam, MA, USA.
Western blot analysis Cells were lysed using the chilled radio-immunoprecipitation assay RIPA buffer, containing mM NaCl, 1. Ethics statement All experimental protocols were approved by the Faculty of Pharmaceutical Sciences, Chulalongkorn University Animal Care and Use Committee, Bangkok, Thailand Number METABRIC analysis The COL17A1 mRNA expression in primary breast cancer and patient clinical data from METABRIC study [ 18 ] were retrieved from cBioPortal [ 45 ].
Statistical analysis All data were reported as mean ± standard deviation. Supporting information. S1 Fig. Tetracycline-regulated lentiviral expression system.
s PDF. S2 Fig. S3 Fig. Analysis of cell death. S4 Fig. Mammary fad pad xenograft. S1 Raw images. Acknowledgments The authors would like to thank the Pharmaceutical Research Instrument Center of the Faculty of Pharmaceutical Sciences at Chulalongkorn University. References 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A.
Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in countries. CA Cancer J Clin. Makki J. Diversity of Breast Carcinoma: Histological Subtypes and Clinical Relevance.
Clin Med Insights Pathol. pmid; PubMed Central PMCID: PMC Fang M, Yuan J, Peng C, Li Y. Collagen as a double-edged sword in tumor progression.
Tumour Biol. Page-McCaw A, Ewald AJ, Werb Z. Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol. Hieda Y, Nishizawa Y, Uematsu J, Owaribe K. Identification of a new hemidesmosomal protein, HD1: a major, high molecular mass component of isolated hemidesmosomes.
J Cell Biol. Fontao L, Stutzmann J, Gendry P, Launay JF. Regulation of the type II hemidesmosomal plaque assembly in intestinal epithelial cells. Exp Cell Res. Borradori L, Sonnenberg A. Structure and function of hemidesmosomes: more than simple adhesion complexes.
J Invest Dermatol. Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.
Franzke CW, Tasanen K, Schacke H, Zhou Z, Tryggvason K, Mauch C, et al. Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs. EMBO J. Nishimura M, Nishie W, Shirafuji Y, Shinkuma S, Natsuga K, Nakamura H, et al. Extracellular cleavage of collagen XVII is essential for correct cutaneous basement membrane formation.
Human Molecular Genetics. Winsey S, Lonie L, Allen J, Bunce M, Marshall SE, Wojnarowska F. Genetic variation in COL17A1 and the development of bullous pemphigoid. Exp Dermatol. Tabatabaei-Panah PS, Moravvej H, Alirajab M, Arghand F, Babaei H, Didehvar E, et al. COL17A1 gene polymorphisms are frequent in bullous pemphigoid.
J Eur Acad Dermatol Venereol. Moilanen JM, Kokkonen N, Loffek S, Vayrynen JP, Syvaniemi E, Hurskainen T, et al. Collagen XVII expression correlates with the invasion and metastasis of colorectal cancer.
Hum Pathol. Moilanen JM, Loffek S, Kokkonen N, Salo S, Vayrynen JP, Hurskainen T, et al. Significant Role of Collagen XVII And Integrin beta4 in Migration and Invasion of The Less Aggressive Squamous Cell Carcinoma Cells.
Sci Rep. Thangavelu PU, Krenacs T, Dray E, Duijf PH. In epithelial cancers, aberrant COL17A1 promoter methylation predicts its misexpression and increased invasion.
Clin Epigenetics. Yodsurang V, Tanikawa C, Miyamoto T, Lo PHY, Hirata M, Matsuda K. Identification of a novel p53 target, COL17A1, that inhibits breast cancer cell migration and invasion. Lee JJ, Loh K, Yap Y-S. Cancer Biol Med. Pereira B, Chin SF, Rueda OM, Vollan HK, Provenzano E, Bardwell HA, et al.
The somatic mutation profiles of 2, breast cancers refines their genomic and transcriptomic landscapes. Nat Commun. Franzke CW, Tasanen K, Borradori L, Huotari V, Bruckner-Tuderman L. J Biol Chem. Liu CC, Lin JH, Hsu TW, Hsu JW, Chang JW, Su K, et al. Geiger T, Madden SF, Gallagher WM, Cox J, Mann M.
Proteomic Portrait of Human Breast Cancer Progression Identifies Novel Prognostic Markers. Cancer Res. Watanabe M, Natsuga K, Nishie W, Kobayashi Y, Donati G, Suzuki S, et al.
Type XVII collagen coordinates proliferation in the interfollicular epidermis. Nicholson KM, Anderson NG. Cell Signal. Ther Adv Med Oncol. Jacków J, Löffek S, Nyström A, Bruckner-Tuderman L, Franzke CW. Collagen XVII Shedding Suppresses Re-Epithelialization by Directing Keratinocyte Migration and Dampening mTOR Signaling.
Löffek S, Hurskainen T, Jackow J, Sigloch FC, Schilling O, Tasanen K, et al. PloS one. Chavez KJ, Garimella SV, Lipkowitz S. Triple negative breast cancer cell lines: one tool in the search for better treatment of triple negative breast cancer. In brief, 5, EO cells were seeded into well plates, treated with 0, 25, 50 and µM Eupafolin, and incubated at 37°C for 24, 36 and 48 h.
Then, 10 µl CCK-8 solution was added to each well and the cells were incubated for 1. A microplate reader was used to measure the absorbance at nm TECAN.
After drug treatment for 24 h, cells were digested with trypsin without EDTA, collected, centrifuged at × g for 5 min at 37°C, resuspended in pre-cooled PBS, centrifuged at × g for 5 min to discard the supernatant, resuspended in µl 1X binding buffer; 5 µl sample was mixed with FITC-Annexin V.
Then, 5 µl propidium iodide PI staining solution was added to µl 1X binding buffer 5 min prior to detection. Flow cytometry was performed using FACS Thermo Fisher Scientific, Inc. Next, cells were resuspended in µl 1X PI solution Baihao for 30 min at 37°C.
Flow cytometry analysis was performed using FACS Thermo Fisher Scientific, Inc. The collected data were analyzed using ModFit LT version 2. to determine cell cycle distribution. Using a sterile µl plastic pipette, a wound was created; the cell debris was removed by washing with PBS and images were captured using an inverted light microscope Olympus Corporation with a digital camera magnification, ×80 at 0 h.
A total of 3 ml of FBS-free medium was added per well; then, 25, 50 and µM Eupafolin was added for 24 h.
Cells were washed with PBS and images were obtained using microscopy. Healing areas were analyzed using ImageJ software version 1. The experiment was performed at least in triplicate. A Transwell Corning, Inc. assay was performed to determine cell migration and invasion abilities. A volume of µl of the cell suspension was added to the upper chamber, and the transwells were placed in an incubator for 24 h.
Next, the cells were removed and the medium was aspirated. The transwell was placed at room temperature and cells were fixed for 20—30 min.
The fixative solution was removed and cells were stained at 37°C using 0. Then, the cells were washed three times with PBS to remove the unbound crystal violet.
The upper surface of the chamber was gently wiped with a cotton swab. Excess crystal violet was removed prior to microscopy.
After drying, five fields were randomly selected and the cells were observed and counted under an inverted light microscope with a digital camera magnification, × According to the instructions provided with the kit, TRIzol reagent Ruan was used to extract total RNA, and cDNA was generated using the FastKing RT kit Tiangen Biotech Co.
The primers used were obtained from NCBI. A real-time fluorescent quantitative PCR detection system Eppendorf was used to perform the RT-qPCR reactions using SYBR-Green Tiangen Biotech Co. and a total of 20 µl reaction mixture.
The qPCR reaction conditions were as follows: Initial denaturation for 15 min at 95°C, followed by 40 cycles of denaturation for 10 sec at 95°C, annealing for 30 sec at 60°C and extension for 20 sec at 72°C.
for 30 min. Then, the BCA method was used to determine the protein concentration: 5X loading buffer was added and proteins were denatured at 95°C by boiling in a water bath for 10 min.
Next, the membranes were incubated with primary antibodies directed against Bcl-2 cat. at 4°C overnight. Subsequently, membranes were washed three times with PBS, then incubated with a corresponding horseradish peroxidase-conjugated secondary antibody ,; cat.
A; Beyotime Institute of Biotechnology for 1. Then, protein bands were visualized using an enhanced chemiluminescence assay kit Beyotime Institute of Biotechnology and photographed using an imaging system Tanon.
Finally, data were analyzed using ImageJ software version 1. The data are presented as the mean ± SD and were analyzed using SPSS v.
Differences between multiple groups were analyzed using one-way ANOVA followed by Dunnett's post hoc test. EO cells were treated with Eupafolin for 24, 36 and 48 h to study the effects of the compound Fig. The results of the CCK-8 assay indicated that Eupafolin inhibited the viability of EO cells, and the inhibitory effect was proportional to the treatment time and dose Fig.
Eupafolin decreases the proliferation of EO cells. A Molecular structure of Eupafolin. B EO cells were exposed to various concentrations of Eupafolin 0, 25, 50 and µM for different durations, and the cell viability was determined using the Cell Counting Kit-8 assay.
Data are shown as mean ± standard deviation from three independent experiments. The scratch test was used to determine the wound healing time of EO cells.
Furthermore, a Transwell assay was performed to determine the ratio of invasion and migration of EO cells Fig. Results showed that compared with the control cells, Eupafolin significantly decreased the migration and invasion of EO cells Fig. MMP2, MMP9 and VEGF-A are positively associated with the migration ability of tumor cells and can be used as marker genes.
Therefore, their expression following Eupafolin treatment was tested, and the results showed that Eupafolin inhibited MMP2, MMP9 and VEGF-A Fig.
Taken together, these results indicate that Eupafolin prevented further deterioration of breast cancer cells. Eupafolin attenuates the invasion and migration of EO cells. After seeding EO cells in a 6-well plate and treating them with different concentrations of Eupafolin for 24 h, the migration distance of the cells was measured, the proportion of invaded cells was calculated, and RNA was extracted from cells to determine the expression of associated genes using RT-qPCR.
A and B 24 h after Eupafolin treatment, the healing effect of EO cells was determined using the scratch test. C The ratio of invasion and migration of EO cells was further determined using a transwell assay.
D EO cells were harvested at 24 h post treatment for RT-qPCR to determine the mRNA expression of MMP2, MMP9 and VEGF-A. Data are expressed as the mean ± SD. MMP, matrix metallopeptidase; RT-qPCR, reverse transcription-quantitative PCR; VEGF, vascular endothelial growth factor.
Next, whether Eupafolin could induce apoptosis in breast cancer cells was tested. Flow cytometry was used to analyze the apoptosis ratio of EO cells following treatment with Eupafolin.
To further understand the specific mechanism of Eupafolin in causing apoptosis in EO cells, the expression of apoptosis-associated proteins was evaluated using western blotting. The protein levels of cleaved caspase 3 and Bax were increased, whereas Bcl-2 protein levels were decreased Fig.
Eupafolin induces apoptosis in EO cells. A EO cells were treated with 25, 50 and µM Eupafolin for 24 h. Cells were then digested and the proportion of apoptosis of breast cancer cells was determined using flow cytometry. B After extraction and treatment, the levels of cleaved caspase 3, Bax, and Bcl-2 proteins were determined using western blotting.
C Western blots were analyzed using ImageJ software and compared with those of the control group. Data are representative of three independent experiments and expressed as the mean ± SD.
After staining with PI, cell cycle analysis of EO cells treated with various concentrations of Eupafolin was performed using flow cytometry.
In addition, RT-qPCR was used to determine the mRNA levels of cycle-associated genes. The results showed that Eupafolin inhibited the expression of cyclin D1, CDK4 and CDK6 mRNA Fig.
A Flow chart showing the distribution of the proportions of different cell cycles. C Extracted RNA was used to determine the mRNA levels of cell cycle-associated genes, cyclin D1, CDK4 and CDK6. Data represent the results of three experiments and are expressed as the mean ± SD. The aforementioned data indicated that Eupafolin had an impact on the proliferation and apoptosis of EO cells.
Therefore, whether Eupafolin affected EO cells through this pathway was tested. Results from western blotting showed that Eupafolin significantly decreased protein levels of p-PI3K, p-Akt and p-mTOR. A EO cells were treated with 25, 50 and µM Eupafolin for 24 h; the proteins were extracted and the associated protein phosphorylation p -PI3K, PI3K, p-Akt, Akt, mTOR and p-mTOR were detected.
B Western blots were analyzed using ImageJ software. Biochemical and morphological alterations induced in mitochondria by lauryl gallate.
A Comparative analysis of Bcl-2 levels in untreated MCF7, MCF7 ADR and MDA-MB cells. Values of Bcl-2 levels after being normalized to the levels of α-tubulin are shown below each blot. B Effect of lauryl gallate treatment on Bcl-2 expression.
After lysis, 30 μg of total cell lysate were separated on SDS—PAGE, and Bcl-2 was detected by western blot with monoclonal antibody anti-Bcl-2 Lower panels show the stripped membranes reprobed with an anti-α-tubulin antibody for loading control. Changes in the levels of Bcl-2 after being normalized to the levels of α-tubulin are shown below each blot.
Control samples at 48 h are considered as the unit. C Flow cytometric analyses of mitochondrial functionality. Mitochondrial membrane depolarization is detected as an increase of FL1 and a decrease of FL2 fluorescence emissions. Data are presented as MFC values and represent the mean ± SD of three independent experiments.
D Transmission electron microscopy analysis of mitochondria of MCF7, MCF7 ADR and MDA-MB cells. Both detached and adherent cells were collected, and electron microscopy analysis was performed as in Figure 4C.
Mitochondria of a control MCF7 cell 1 , arrows indicate parallel cristae. Mitochondria of a lauryl gallate-treated MCF7 cell 2 , arrows indicate two organelles with a reduced number of the cristae.
Mitochondria of a lauryl gallate-treated MCF7 ADR cell 3 , arrow indicates a swollen mitochondrion showing loss of cristae.
Mitochondria of a lauryl gallate-treated MDA-MB cell 4 , arrow indicates a highly condensed mitochondrion showing vacuolization of the cristae. As described above, lauryl gallate-induced apoptosis involved reduction of Bcl-2 expression and mitochondrial membrane depolarization and structural modifications.
Furthermore, Bcl-2 expression was higher in MCF7 than in the other two cell lines, suggesting that Bcl-2 could account for the lower sensitivity of MCF7 to lauryl gallate.
To examine this hypothesis, a Bcl-2 overexpressing clone MAB25 derived from transfected MCF7 ADR cells was used Cell viability and annexin V-FITC labeling analyses showed that overexpression of Bcl-2 protected cells from apoptosis. Treatment with lauryl gallate significantly reduced cell viability Figure 6A and induced apoptosis Figure 6B in MAN9 control transfectant clone when compared to MAB25 Bcl-2 overexpressing clone.
When analyzed by transmission electron microscopy, MAN9 showed evident morphological alterations condensed chromatin, vacuolization of cytoplasm after treatment with lauryl gallate 5 μM for 48 h, Figure 6C2 as compared to control cells Figure 6C1.
At higher magnification, dilation of endoplasmic reticulum and nuclear envelope were clearly detectable and mitochondria appeared strongly altered, showing swelling and loss of cristae Figure 6C3.
On the contrary, few treated MAB25 cells showed morphological features of apoptosis, as most of them displayed nuclear and cytoplasmic morphology Figure 6C5 similar to control Figure 6C4. Furthermore, most of mitochondria preserved their ultrastructure despite 48 h of treatment Figure 6C6.
Effect of Bcl-2 and p53 expression on lauryl gallate-induced cytotoxicity. A Analysis of cell viability of Bcl-2 overexpressing clone MAB25 and of control transfectant clone MAN9. Cells were treated with 5 or 10 μM lauryl gallate LG for 72 h and counted as in Figure 1. B Analysis of annexin V binding.
C Transmission electron microscopy analysis of MAN9 and MAB25 clones. Cells were treated with 5 μM lauryl gallate for 48 h and processed for electron microscopy analysis as in Figure 4C. Electron micrographs of MAN9 cells 1, control; 2 and 3, lauryl gallate-treated cells , and MAB25 cells 4, control; 5 and 6, lauryl gallate-treated cells.
Arrows point to dilation of the nuclear envelope. Arrowheads indicate dilation of endoplasmic reticulum ER. N, nucleus; M, mitochondria. D Analysis of cell viability left panel and apoptosis right panel in MCF7-C4 and E6 cell lines, in MDA-MB clones 1 and 8, expressing temperature-sensitive p Analyses were performed as described in panels A and B, respectively.
Since the most sensitive cells were those expressing mutant inactive forms of p53, MCF7 ADR and MDA-MB, we next analyzed the possible contribution of p53 as a target for lauryl gallate effects by two independent approaches. First, we used a MCF7 clone MCF7-E6 expressing E6 protein from human papilloma virus type 16, which induces p53 degradation, therefore expressing lower levels of wild-type p53 than control clone C4 data not shown Second, we generated clones of MDA-MB expressing temperature-sensitive mutant p53Val, which acquires active wild-type conformation at 32°C Clones were tested for increased expression of p21 at 32°C versus 37°C by western blot data not shown , and clones 1 and 8 were selected.
MCF7 and MDA-MB clones were treated as described above to study cytotoxic responses cell viability and apoptosis to lauryl gallate. If wild-type p53 confers a certain degree of resistance, decreasing its expression in MCF7-E6 should make them more sensitive to lauryl gallate than control MCF7-C4.
Although we observed a slightly higher sensitivity to lauryl gallate decreased cell viability and increased apoptosis , when calculations were made adjusting basal values, there were not significant differences between control MCF7-C4 clone and MCF7-E6 Figure 6D. Similarly, the expression of functional p53 in MDA-MB was supposed to reduce sensitivity to lauryl gallate.
However, no significant differences were found between clones expressing pVal at 32 and 37°C Figure 6D. Mitogen-activated protein kinase MAPK signaling pathways are activated in response to several cytotoxic compounds To explore whether lauryl gallate could induce MAPK activation, lysates from cells treated with 5 μM lauryl gallate for 24, 48 and 72 h were subjected to western blot analysis using anti-phospho-MAPKs antibodies to detect their activation.
Concerning other members of the MAPK family, no phosphorylation of p38 kinase was observed, while only a light c-jun N-terminal kinase JNK activation was detected data not shown. As previously shown Figure 3 lauryl gallate caused MCF7 accumulation in G 1 , while in MCF7 ADR and MDA-MB it induced slow-down of cell cycle progression.
MCF7, MCF7 ADR and MDA-MB cells were cultured with 5 μM lauryl gallate LG or DMSO, as a control, for 24, 48 and 72 h. Lower panels show the stripped membranes reprobed with anti-Erk2 antibody for loading control. Cells were treated with 10 μM PD for 2 h prior to addition of 5 μM lauryl gallate LG for 48 h.
For p21 detection, 40 μg of total cell lysate were separated on SDS—PAGE, transferred to a membrane that was then incubated with monoclonal antibody anti-p21 Control samples are considered as the unit. Results shown are representative of three independent experiments.
Cell cycle analyses were performed as in Figure 3A. As observed in Figure 7C , lower panel, cells treated with 10 μM PD for 2 h prior to addition of 5 μM lauryl gallate for 48 h, showed a significant reduction of the percentage of apoptotic cells as compared with lauryl gallate-treated cells.
The effect of lauryl gallate, an antioxidant gallic acid derivative, has been studied on three human breast cancer cell lines: wild-type p53 MCF7, and two non-functional p53 cell lines, MCF7 ADR multidrug-resistant and the highly proliferative and invasive MDA-MB Our results show that lauryl gallate behaved as a potent antiproliferative agent inducing cell cycle alterations and apoptosis in all cell lines tested.
A wide variety of cytotoxic insults result in activation of mechanisms that arrest cell cycle progression at specific checkpoints, presumably to allow cells to repair DNA damage.
This is consistent with G 1 arrest of MCF7 induced by lauryl gallate. However, in MCF7 ADR and MDA-MB, a stable block in G 1 phase was not achieved and cell cycle progressed allowing cells to accumulate damages.
Cell cycle-mediated apoptosis is a common way for cell death upon treatment with anti-tumor agents and inability to activate the apoptotic program confers drug resistance.
Another mechanism to evade the action of cytotoxic agents is to express MDR genes, such as mdr-1 encoding for P-gp 4. For that reason, one of the strategies employed to overcome drug resistance is to inhibit the function of P-gp, allowing resistant cells to accumulate drugs to levels similar to those found in sensitive cells Therefore, it is of an utmost importance to obtain compounds that will not be affected by P-gp excretory action.
Our results demonstrate that lauryl gallate induces strong effects in the multidrug-resistant MCF7 ADR cells, characterized by P-gp overexpression. Similarly, cytofluorimetric assays by doxorubicin accumulation and UIC2 shift demonstrated that lauryl gallate did not interfere with the proper functionality of P-gp when analyzed towards other compounds.
This result is in contrast with recent studies demonstrating that polyphenols from green tea, such as epigallocatechin gallate, inhibited activity of P-gp, increasing intracellular accumulation of P-gp substrates, acting as potential MDR reversal agents when used in combination with antitumoral compounds 40 , The higher complexity of these polyphenols may explain a different behavior from the one we report for lauryl gallate.
Among proteins involved in control of apoptotic pathway, Bcl-2 has been demonstrated to protect cells from both normal and experimentally induced apoptosis Bcl-2 is located in the outer mitochondrial membrane and its apoptosis-regulatory effects are mainly related with mitochondrial structure and function.
This protein prevents opening of mitochondrial permeability transition pores and, subsequently, inhibits mitochondrial membrane depolarization and release of apoptogenic factors to cytosol 29 , The higher Bcl-2 protein levels detected in MCF7 as compared to the other two cell lines could result in a lower sensitivity to lauryl gallate.
This hypothesis was confirmed by electron microscopy and flow cytometry analyses, showing lower functional and morphological alterations in mitochondria of MCF7 cells as compared to those observed in MDA-MB and MCF7 ADR cells. Furthermore, the protective role of Bcl-2 against lauryl gallate-induced apoptosis was clearly demonstrated by higher viability and lower number of cells with morphological apoptotic features shown by MAB25 Bcl-2 overexpressing MCF7 ADR clone as compared to MAN9 control clone.
Similar protective effects of Bcl-2 have been reported in murine B cell lymphoma line Wehi Other studies have demonstrated that a partial resistance of MCF7 against camptothecin and UCN is related to Bcl-2 expression Activation of G 1 checkpoint requires functional p53, which transcriptionally activates downstream genes to induce G 1 cell cycle arrest and, in some cases, apoptosis Mutation of p53 tumor suppressor gene is one of the most common genetic alterations in human cancers, including breast cancer, which induces uncontrolled cell proliferation Wild-type p53 protein has been shown to mediate apoptosis, while mutations or deletions within p53 gene have been associated, in vivo and in vitro , both with resistance to apoptosis by different chemotherapeutic agents in some cell lines and with increased sensitivity in others 44 , Results obtained in MCF7-E6 cells and in MDA-MB pVal suggest that p53 is not a major target for lauryl gallate-induced cytotoxicity.
Among pactivated genes, p21 is a potent inhibitor of cyclin-dependent kinase activities required for progression from G 1 to S phase of the cell cycle Surprisingly, lauryl gallate induced p21 expression in all three cell lines tested.
Increased p21 expression in MCF7 correlated with G 1 arrest, and a delayed cell cycle progression in the other two cell lines. As MCF7 ADR and MDA-MB cells express a mutated p53, induction of p21 by lauryl gallate could be attributed to activation of a pindependent pathway.
Also, its contribution to cell proliferation and survival is well established Phosphorylation of Bcl-2, usually distributed in hypophosphorylated form on the mitochondrial membrane, results in reduction of its anti-apoptotic activity and redistribution from mitochondria to cytoplasm.
In addition, the lower cytotoxicity observed in MCF7 and MAB25 could be in agreement with this theory. Given that these cells expressed elevated levels of Bcl-2, a higher lauryl gallate concentration may be required to obtain the same effect as those observed in MCF7 ADR and MDA-MB with lower levels of Bcl As for activation of other MAPKs, lauryl gallate did not induce p38 phosphorylation and only slightly activated JNK data not shown.
In conclusion, our data indicate that lauryl gallate induced reduction of cell survival and cell cycle alterations in MCF7, MCF7 ADR and MDA-MB cells. An evident p21 upregulation was observed in all three breast cancer cell lines, followed by apoptotic cell death.
Interestingly, a stronger cytotoxic effect was observed on multidrug-resistant MCF7 ADR cells as well as on MDA-MB carrying mutations in the p53 gene, which, in theory, should be more resistant to cytotoxic treatments and apoptosis.
These findings suggest that lauryl gallate might be a good candidate for innovative therapeutic strategies against tumors carrying p53 mutations and resistant to conventional chemotherapeutic agents.
We thank J. Pérez and L. Camilli for the artwork. Authors are grateful to D. Del Bulafo and D. Trisciuoglio for donation of MCF7 ADR MAN9 and MAB25 clones, and together with I. Lazaro-Trueba for their constructive comments. JMP is a member of GEICAM. Google Scholar.
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Journal Article. Inhibition of proliferation and induction of apoptosis in human breast cancer cells by lauryl gallate. Annarica Calcabrini , Annarica Calcabrini.
Oxford Academic. José Manuel García-Martínez. Lorena González. Mercedes Julián Tendero. María Teresa Agulló Ortuño. Pasqualina Crateri. Abelardo Lopez-Rivas. Giuseppe Arancia. Pedro González-Porqué. Revision received:. PDF Split View Views. Select Format Select format. ris Mendeley, Papers, Zotero.
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