Pulsed Electromagnetic Field Enhances Caffeic Acid Phenethyl Ester-induced Death of MCF-7 Breast Cancer Cells.

BACKGROUND/AIM: Caffeic acid phenethyl ester (CAPE) exerts anticancer effects against several cancer types, including breast cancer. Pulsed electromagnetic field (PEMF) improves the efficiency of some chemotherapeutic drugs. In this study, we examined the effects of PEMF stimulation on the anticancer activity of CAPE in MCF-7 breast cancer cells and the underlying signal transduction pathways. MATERIALS AND METHODS: MCF-7 cells were seeded and incubated for 24 h. Each of the drugs (5-fluorouracil, paclitaxel, gefitinib, or CAPE) was added to the cells on day 0. Then, cells were immediately stimulated with a 60-min PEMF session thrice a day (with 4-h interval between sessions) for 1-3 days. Cell death and viability were assessed by flow cytometry and trypan blue dye exclusion assay. Molecular mechanisms involved in cell death were confirmed by western blot assay. RESULTS: Compared with treatment with CAPE alone, co-treatment with CAPE and PEMF more strongly reduced the viability of MCF-7 cells, further increased the percentage of the sub-G1 population, poly (ADP-ribose) polymerase (PARP) cleavage, activation of apoptotic caspases, up-regulation of pro-apoptotic proteins, such as Fas cell surface death receptor (FAS) and BCL2 associated X, apoptosis regulator (BAX), and reduced the expression of anti-apoptotic proteins, such as BCL-2 apoptosis regulator (BCL-2), MCL-1 apoptosis regulator, BCL-2 family member (MCL-1), and survivin. PEMF stimulation also increased CAPE-induced phosphorylation of p53, and inhibition of p53 partially restored the PEMF-reduced viability of CAPE-treated MCF-7 cells. CONCLUSION: PEMF stimulation enhanced CAPE-induced cell death by activating p53, which regulates the expression of apoptosis-related molecules, subsequently activating the caspase-dependent apoptotic pathway in MCF-7 cells, suggesting that PEMF can be utilized as an adjuvant to enhance the effect of CAPE on breast cancer cells.

Parkin Enhances Gefitinib-induced Anoikis in HeLa Cervical Cancer Cells.

BACKGROUND/AIM: Gefitinib exhibits anticancer activity against cervical cancer cells via anoikis, a type of apoptosis induced by cell detachment from the extracellular matrix. Previous studies have reported that Parkin expression affects the efficacy of anticancer drugs. However, the impact of Parkin expression on the therapeutic effects of gefitinib in human cervical cancer remains unclear. Thus, this study aimed to evaluate whether Parkin over-expression improves the therapeutic effects of gefitinib against HeLa cervical cancer cells. MATERIALS AND METHODS: Cell viability and apoptotic death of HeLa cells were measured by trypan blue dye exclusion assay and flow cytometry. Cell detachment, adhesion, spreading, and cell-cell interaction were observed by inverted microscopy. Alteration of adhesion-related molecules was evaluated by confocal microscopy and western blot assay. RESULTS: Parkin expression potentiated gefitinib-induced cell detachment by affecting the organization of the actin cytoskeleton. In addition, Parkin expression induced a further reduction in the reattachment of and interaction between detached cells. The therapeutic efficacy of low-dose gefitinib combined with Parkin expression was equivalent to that of high-dose gefitinib alone. CONCLUSION: Parkin expression promotes gefitinib-induced anoikis, consequently increasing the efficacy of gefitinib against HeLa human cervical cancer cells. Based on our results, we propose that Parkin can be used to increase the anti-cancer effect of gefitinib on cervical cancer cells.

Gefitinib induces anoikis in cervical cancer cells.

Gefitinib exerts anticancer effects on various types of cancer, such as lung, ovarian, breast, and colon cancers. However, the therapeutic effects of gefitinib on cervical cancer and the underlying mechanisms remain unclear. Thus, this study aimed to explore whether gefitinib can be used to treat cervical cancer and elucidate the underlying mechanisms. Results showed that gefitinib induced a caspase-dependent apoptosis of HeLa cells, which consequently became round and detached from the surface of the culture plate. Gefitinib induced the reorganization of actin cytoskeleton and downregulated the expression of p-FAK, integrin ß1 and E-cadherin, which are important in cell-extracellular matrix adhesion and cell-cell interaction, respectively. Moreover, gefitinib hindered cell reattachment and spreading and suppressed interactions between detached cells in suspension, leading to poly (ADP-ribose) polymerase cleavage, a hallmark of apoptosis. It also induced detachment-induced apoptosis (anoikis) in C33A cells, another cervical cancer cell line. Taken together, these results suggest that gefitinib triggers anoikis in cervical cancer cells. Our findings may serve as a basis for broadening the range of anticancer drugs used to treat cervical cancer. [BMB Reports 2024; 57(2): 104-109].

Cellular subpopulations identified using an ensemble average of multiple dielectrophoresis measurements.

While the average value measurement approach can successfully analyze and predict the general behavior and biophysical properties of an isogenic cell population, it fails when significant differences among individual cells are generated in the population by intracellular changes such as the cell cycle, or different cellular responses to certain stimuli. Detecting such single-cell differences in a cell population has remained elusive. Here, we describe an easy-to-implement and generalizable platform that measures the dielectrophoretic cross-over frequency of individual cells by decreasing measurement noise with a stochastic method and computing ensemble average statistics. This platform enables multiple, real-time, label-free detection of individual cells with significant dielectric variations over time within an isogenic cell population. Using a stochastic method in combination with the platform, we distinguished cell subpopulations from a mixture of drug-untreated and -treated isogenic cells. Furthermore, we demonstrate that our platform can identify drug-treated isogenic cells with different recovery rates.

Tumor suppressor Parkin induces p53-mediated cell cycle arrest in human lung and colorectal cancer cells.

Dysregulation of the E3 ubiquitin ligase Parkin has been linked to various human cancers, indicating that Parkin is a tumor suppressor protein. However, the mechanisms of action of Parkin remain unclear to date. Thus, we aimed to elucidate the mechanisms of action of Parkin as a tumor suppressor in human lung and colorectal cancer cells. Results showed that Parkin overexpression reduced the viability of A549 human lung cancer cells by inducing G2/M cell cycle arrest. In addition, Parkin caused DNA damage and ATM (Ataxia telangiectasia mutated) activation, which subsequently led to p53 activation. It also induced the p53-mediated upregulation of p21 and downregulation of cyclin B1. Moreover, Parkin suppressed the proliferation of HCT-15 human colorectal cancer cells by a mechanism similar to that in A549 lung cancer cells. Taken together, our results suggest that the tumor-suppressive effects of Parkin on lung and colorectal cancer cells are mediated by DNA damage/p53 activation/cyclin B1 reduction/cell cycle arrest. [BMB Reports 2023; 56(10): 557-562].

Triglyceride induces DNA damage leading to monocyte death by activating caspase-2 and caspase-8.

Monocytes are peripheral leukocytes that function in innate immunity. Excessive triglyceride (TG) accumulation causes monocyte death and thus can compromise innate immunity. However, the mechanisms by which TG mediates monocyte death remain unclear to date. Thus, this study aimed to elucidate the mechanisms by which TG induces monocyte death. Results showed that TG induced monocyte death by activating caspase-3/7 and promoting poly (ADP-ribose) polymerase (PARP) cleavage. In addition, TG induced DNA damage and activated the ataxia telangiectasia mutated (ATM)/checkpoint kinase 2 and ATM-and Rad3-related (ATR)/checkpoint kinase 1 pathways, leading to the cell death. Furthermore, TG-induced DNA damage and monocyte death were mediated by caspase-2 and -8, and caspase-8 acted as an upstream molecule of caspase-2. Taken together, these results suggest that TG-induced monocyte death is mediated via the caspase-8/caspase-2/DNA damage/executioner caspase/PARP pathways. [BMB Reports 2023; 56(3): 166-171].

On-Chip Single-Cell Bioelectrical Analysis for Identification of Cell Electrical Phenotyping in Response to Sequential Electric Signal Modulation.

In recent years, an interesting biomarker called membrane breakdown voltage has been examined using artificial planar lipid bilayers. Even though they have great potential to identify cell electrical phenotyping for distinguishing similar cell lines or cells under different physiological conditions, the biomarker has not been evaluated in the context of living cell electrical phenotyping. Herein, we present a single-cell analysis platform to continuously measure the electric response in a large number of cells in parallel using electric frequency and voltage variables. Using this platform, we measured the direction of cell displacement and transparent cell image alteration as electric polarization of the cell responds to signal modulation, extracting the dielectrophoretic crossover frequency and membrane breakdown voltage for each cell, and utilizing the measurement results in the same spatiotemporal environment. We developed paired parameters using the dielectrophoretic crossover frequency and membrane breakdown voltage for each cell and evaluated the paired parameter efficiency concerning the identification of two different breast cancer cells and cell drug response. Moreover, we showed that the platform was able to identify cell electrical phenotyping, which was generated by subtle changes in cholesterol depletion-induced cell membrane integrity disruption when the paired parameter was used. Our platform introduced in this paper is extremely useful for facilitating more accurate and efficient evaluation of cell electrical phenotyping in a variety of applications, such as cell biology and drug discovery.

Pulsed electromagnetic field potentiates etoposide-induced MCF-7 cell death.

Etoposide is a chemotherapeutic medication used to treat various types of cancer, including breast cancer. It is established that pulsed electromagnetic field (PEMF) therapy can enhance the effects of anti-cancer chemotherapeutic agents. In this study, we investigated whether PEMFs influence the anti-cancer effects of etoposide in MCF-7 cells and determined the signal pathways affected by PEMFs. We observed that co-treatment with etoposide and PEMFs led to a decrease in viable cells compared with cells solely treated with etoposide. PEMFs elevated the etoposide- induced PARP cleavage and caspase-7/9 activation and enhanced the etoposide-induced down-regulation of survivin and up-regulation of Bax. PEMF also increased the etoposideinduced activation of DNA damage-related molecules. In addition, the reactive oxygen species (ROS) level was slightly elevated during etoposide treatment and significantly increased during co-treatment with etoposide and PEMF. Moreover, treatment with ROS scavenger restored the PEMF-induced decrease in cell viability in etoposide-treated MCF-7 cells. These results combined indicate that PEMFs enhance etoposide-induced cell death by increasing ROS induction-DNA damage-caspase-dependent apoptosis. [BMB Reports 2022; 55(3): 148-153].

A Novel Method for Effective Cell Segmentation and Tracking in Phase Contrast Microscopic Images.

Cell migration plays an important role in the identification of various diseases and physiological phenomena in living organisms, such as cancer metastasis, nerve development, immune function, wound healing, and embryo formulation and development. The study of cell migration with a real-time microscope generally takes several hours and involves analysis of the movement characteristics by tracking the positions of cells at each time interval in the images of the observed cells. Morphological analysis considers the shapes of the cells, and a phase contrast microscope is used to observe the shape clearly. Therefore, we developed a segmentation and tracking method to perform a kinetic analysis by considering the morphological transformation of cells. The main features of the algorithm are noise reduction using a block-matching 3D filtering method, k-means clustering to mitigate the halo signal that interferes with cell segmentation, and the detection of cell boundaries via active contours, which is an excellent way to detect boundaries. The reliability of the algorithm developed in this study was verified using a comparison with the manual tracking results. In addition, the segmentation results were compared to our method with unsupervised state-of-the-art methods to verify the proposed segmentation process. As a result of the study, the proposed method had a lower error of less than 40% compared to the conventional active contour method.

Activation of pannexin-1 mediates triglyceride-induced macrophage cell death.

The accumulation of triglycerides (TGs) in macrophages induces cell death, a risk factor in the pathogenesis of atherosclerosis. We had previously reported that TG-induced macrophage death is triggered by caspase-1 and -2, therefore we investigated the mechanism underlying this phenomenon. We found that potassium efflux is increased in TG-treated THP-1 macrophages and that the inhibition of potassium efflux blocks TG-induced cell death as well as caspase-1 and -2 activation. Furthermore, reducing ATP concentration (known to induce potassium efflux), restored cell viability and caspase-1 and -2 activity. The activation of pannexin-1 (a channel that releases ATP), was increased after TG treatment in THP-1 macrophages. Inhibition of pannexin-1 activity using its inhibitor, probenecid, recovered cell viability and blocked the activation of caspase-1 and -2 in TG-treated macrophages. These results suggest that TG-induced THP-1 macrophage cell death is induced via pannexin- 1 activation, which increases extracellular ATP, leading to an increase in potassium efflux. [BMB Reports 2020; 53(11): 588-593].

Variable Membrane Dielectric Polarization Characteristic in Individual Live Cells.

Investigation of the dielectric properties of cell membranes plays an important role in understanding the biological activities that sustain cellular life and realize cellular functionalities. Herein, the variable dielectric polarization characteristics of cell membranes are reported. In controlling the dielectric polarization of a cell using dielectrophoresis force spectroscopy, different cellular crossover frequencies were observed by modulating both the direction and sweep rate of the frequency. The crossover frequencies were used for the extraction of the variable capacitance, which is involved in the dielectric polarization across the cell membranes. In addition, this variable phenomenon was investigated by examining cells whose membranes were cholesterol-depleted with methyl-ß-cyclodextrin, which verified a strong correlation between the variable dielectric polarization characteristics and membrane composition changes. This study presented the dielectric polarization properties in live cells' membranes that can be modified by the regulation of external stimuli and provided a powerful platform to explore cellular membrane dielectric polarization.

A novel automatic segmentation and tracking method to measure cellular dielectrophoretic mobility from individual cell trajectories for high throughput assay.

BACKGROUND AND OBJECTIVE: The dielectrophoresis (DEP) technique is increasingly being recognised as a potentially valuable tool for non-contact manipulation of numerous cells as well as for biological single cell analysis with non-invasive characterisation of a cell's electrical properties. Several studies have attempted to track multiple cells to characterise their cellular DEP mobility. However, they encountered difficulties in simultaneously tracking the movement of a large number of individual cells in a bright-field image sequence because of interference from the background electrode pattern. Consequently, this present study aims to develop an automatic system for imaging-based characterisation of cellular DEP mobility, which enables the simultaneous tracking of several hundred of cells inside a microfluidic device. METHODS: The proposed method for segmentation and tracking of cells consists of two main stages: pre-processing and particle centre localisation. In the pre-processing stage, background subtraction and contrast enhancement were performed to distinguish the cell region from the background image. In the particle centre localisation stage, the unmarked cell was automatically detected via graph-cut algorithm-based K-means clustering. RESULTS: Our algorithm enabled segmentation and tracking of numerous Michigan Cancer Foundation-7 (MCF-7) cell trajectories while the DEP force was oscillated between positive and negative. The cell tracking accuracy and cell count capability was at least 90% of the total number of cells with the newly developed algorithm. In addition, the cross-over frequency was measured by analysing the segmented and tracked trajectory data of the cellular movements caused by the positive and negative DEP force. The measured cross-over frequency was compared with previous results. The multi-cellular movements investigation based on the measured cross-over frequency was repeated until the viability of cells was unchanged in the same environment as in a microfluidic device. The results were statistically consistent, indicating that the developed algorithm was reliable for the investigation of DEP cellular mobility. CONCLUSION: This study developed a powerful platform to simultaneously measure the DEP-induced trajectories of numerous cells, and to investigate in a robust, efficient, and accurate manner the DEP properties at both the single cell and cell ensemble level.

Hypoxia-induced apoptosis of astrocytes is mediated by reduction of Dicer and activation of caspase-1.

Hypoxia is a condition in which the whole body or a region of the body is deprived of oxygen supply. The brain is very sensitive to the lack of oxygen and cerebral hypoxia can rapidly cause severe brain damage. Astrocytes are essential for the survival and function of neurons. Therefore, protecting astrocytes against cell death is one of the main therapeutic strategies for treating hypoxia. Hence, the mechanism of hypoxia-induced astrocytic cell death should be fully elucidated. In this study, astrocytes were exposed to hypoxic conditions using a hypoxia work station or the hypoxia mimetic agent cobalt chloride (CoCl2 ). Both the hypoxic gas mixture (1% O2 ) and chemical hypoxia-induced apoptotic cell death in T98G glioblastoma cells and mouse primary astrocytes. Reactive oxygen species were generated in response to the hypoxia-mediated activation of caspase-1. Active caspase-1 induced the classical caspase-dependent apoptosis of astrocytes. In addition, the microRNA processing enzyme Dicer was cleaved by caspase-3 during hypoxia. Knockdown of Dicer using antisense oligonucleotides induced apoptosis of T98G cells. Taken together, these results suggest that astrocytic cell death during hypoxia is mediated by the reactive oxygen species/caspase-1/classical caspase-dependent apoptotic pathway. In addition, the decrease in Dicer levels by active caspase-3 amplifies this apoptotic pathway via a positive feedback loop. These findings may provide a new target for therapeutic interventions in cerebral hypoxia.

Non-Linear Cellular Dielectrophoretic Behavior Characterization Using Dielectrophoretic Tweezers-Based Force Spectroscopy inside a Microfluidic Device.

Characterization of cellular dielectrophoretic (DEP) behaviors, when cells are exposed to an alternating current (AC) electric field of varying frequency, is fundamentally important to many applications using dielectrophoresis. However, to date, that characterization has been performed with monotonically increasing or decreasing frequency, not with successive increases and decreases, even though cells might behave differently with those frequency modulations due to the nonlinear cellular electrodynamic responses reported in previous works. In this report, we present a method to trace the behaviors of numerous cells simultaneously at the single-cell level in a simple, robust manner using dielectrophoretic tweezers-based force spectroscopy. Using this method, the behaviors of more than 150 cells were traced in a single environment at the same time, while a modulated DEP force acted upon them, resulting in characterization of nonlinear DEP cellular behaviors and generation of different cross-over frequencies in living cells by modulating the DEP force. This study demonstrated that living cells can have non-linear di-polarized responses depending on the modulation direction of the applied frequency as well as providing a simple and reliable platform from which to measure a cellular cross-over frequency and characterize its nonlinear property.

Visualization of MMP-2 Activity Using Dual-Probe Nanoparticles to Detect Potential Metastatic Cancer Cells.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes capable of degrading extracellular matrix components. Previous studies have shown that the upregulation of MMP-2 is closely related to metastatic cancers. While Western blotting, zymography, and Enzyme-Linked Immunosorbent Assays (ELISA) can be used to measure the amount of MMP-2 activity, it is not possible to visualize the dynamic MMP-2 activities of cancer cells using these techniques. In this study, MMP-2-activated poly(lactic-co-glycolic acid) with polyethylenimine (MMP-2-PLGA-PEI) nanoparticles were developed to visualize time-dependent MMP-2 activities. The MMP-2-PLGA-PEI nanoparticles contain MMP-2-activated probes that were detectable via fluorescence microscopy only in the presence of MMP-2 activity, while the Rhodamine-based probes in the nanoparticles were used to continuously visualize the location of the nanoparticles. This approach allowed us to visualize MMP-2 activities in cancer cells and their microenvironment. Our results showed that the MMP-2-PLGA-PEI nanoparticles were able to distinguish between MMP-2-positive (HaCat) and MMP-2-negative (MCF-7) cells. While the MMP-2-PLGA-PEI nanoparticles gave fluorescent signals recovered by active recombinant MMP-2, there was no signal recovery in the presence of an MMP-2 inhibitor. In conclusion, MMP-2-PLGA-PEI nanoparticles are an effective tool to visualize dynamic MMP-2 activities of potential metastatic cancer cells.

Regulatory Effects of Black Rice Extract on Helicobacter pylori Infection-Induced Apoptosis.

SCOPE: Black rice extract (BRE) contains cyanidin 3-O-glucoside (C3G), an anthocyanin, as the major component. In this study, we found that BRE inhibits the mRNA and protein expression of genes encoding cytotoxin-associated protein A (cagA) and vacuolating protein A (vacA) in Helicobacter pylori 60190 strain. METHODS AND RESULTS: We performed RT-PCR and western blotting to show that BRE inhibits the mRNA and protein expression of SecA. Because SecA is involved in VacA export in bacteria, our result suggests a positive correlation between BRE-induced inhibition of secA expression and VacA secretion. Further, we perform MTT assay and flow cytometry to show that BRE decreases the apoptosis of H. pylori-infected KATO III cells. Finally, we perform western blotting to show that the cell-protective effect of BRE is associated with decreased levels of active proapoptotic proteins caspases and PARP and increased levels of antiapoptotic proteins survivin and XIAP in H. pylori-infected cells. CONCLUSION: Thus, our results indicate that BRE acts as a potent inhibitor of the biogenesis of H. pylori virulence proteins and decreases the apoptosis of H. pylori-infected cells. Moreover, our results suggest that BRE can be used to exert beneficial effects in patients with gastroduodenal diseases caused by H. pylori.

Caspase-2 mediates triglyceride (TG)-induced macrophage cell death.

Triglyceride (TG) accumulation causes macrophage cell death, which affects the development of atherosclerosis. Here, we examined whether caspase-2 is implicated in TG-induced macrophage cell death. We found that caspase-2 activity is increased in TG-treated THP-1 macrophages, and that inhibition of caspase-2 activity drastically inhibits TG-induced cell death. We previously reported that TG-induced macrophage cell death is triggered by caspase-1, and thus investigated the relationship between caspase-2 and caspase-1 in TG-induced macrophage cell death. Inhibition of caspase-2 activity decreased caspase-1 activity in TG-treated macrophages. However, caspase-1 inhibition did not affect caspase-2 activity, suggesting that caspase-2 is upstream of caspase-1. Furthermore, we found that TG induces activation of caspase-3, -7, -8, and -9, as well as cleavage of PARP. Inhibition of caspase-2 and -1 decreased TG-induced caspase-3, -7, -8, and -9 activation and PARP cleavage. Taken together, these results suggest that TG-induced macrophage cell death is mediated via the caspase-2/caspase-1/apoptotic caspases/PARP pathways. [BMB Reports 2017; 50(10): 510-515].

Amplification of distinct α-synuclein fibril conformers through protein misfolding cyclic amplification.

Amyloid fibril formation has been implicated in the pathogenesis of neurodegenerative diseases. Fibrillation generates numerous conformers. Presumably, the conformers may possess specific biological properties, thus providing a biochemical framework for strains of prions. However, the precise relationship between various fibril conformers and their pathogenic functions has not been determined because of limited accessibility to adequate amounts of fibrils from tissue samples. α-Synuclein is one such protein, and it has been implicated in Parkinson disease. Using a technique known as protein misfolding cyclic amplification, originally developed for amplifying prions, we established a procedure through which the amplification of α-synuclein fibrils is possible. With a trace amount of seeds, we succeeded in amplifying α-synuclein fibrils. The replication of the seeds was faithful in terms of conformation even after multiple rounds of cyclic amplification. Moreover, two transgenic mouse strains each representing a distinct synucleinopathy were used to investigate different conformers by using this technique. The amplified α-synuclein fibrils derived from the tissue extracts of these two strains led to the production of two different fibril conformers with distinct proteinase K digestion profiles. Together, our results demonstrated that a trace amount of α-synuclein fibrils in tissue extracts could be amplified with their conformations conserved. This procedure should be useful in amplifying α-synuclein fibrils from the brains and body fluids of patients afflicted with synucleinopathies and may serve as a potential diagnostic tool for Parkinson disease and other synucleinopathies.

Parkin induces G2/M cell cycle arrest in TNF-α-treated HeLa cells.

Parkin is a known tumor suppressor. However, the mechanism by which parkin acts as a tumor suppressor remains to be fully elucidated. Previously, we reported that parkin expression induces caspase-dependent apoptotic cell death in TNF-α-treated HeLa cells. However, at that time, we did not consider the involvement of parkin in cell cycle control. In the current study, we investigated whether parkin is involved in cell cycle regulation and suppression of cancer cell growth. In our cell cycle analyses, parkin expression induced G2/M cell cycle arrest in TNF-α-treated HeLa cells. To elucidate the mechanism(s) by which parkin induces this G2/M arrest, we analyzed cell cycle regulatory molecules involved in the G2/M transition. Parkin expression induced CDC2 phosphorylation which is known to inhibit CDC2 activity and cause G2/M arrest. Cyclin B1, which is degraded during the mitotic transition, accumulated in response to parkin expression, thereby indicating parkin-induced G2/M arrest. Next, we established that Myt1, which is known to phosphorylate and inhibit CDC2, increased following parkin expression. In addition, we found that parkin also induces increased Myt1 expression, G2/M arrest, and reduced cell viability in TNF-α-treated HCT15 cells. Furthermore, knockdown of parkin expression by parkin-specific siRNA decreased Myt1 expression and phosphorylation of CDC2 and resulted in recovered cell viability. These results suggest that parkin acts as a crucial molecule causing cell cycle arrest in G2/M, thereby suppressing tumor cell growth.

Parkin induces upregulation of 40S ribosomal protein SA and posttranslational modification of cytokeratins 8 and 18 in human cervical cancer cells.

Parkin was originally identified as a protein associated with Parkinson's disease. Recently, numerous research studies have suggested that parkin acts as a tumor suppressor. In accordance with these studies, we previously reported that overexpression of parkin in HeLa cells induced growth inhibition. To elucidate possible mechanisms by which parkin may inhibit cell growth, HeLa cells were infected with adenoviruses expressing either the parkin gene or adenovirus alone for 72 h and a total proteomic analysis was performed using 2-D gel electrophoresis followed by LC-MS/MS. We identified three proteins whose expression changed between the two groups: the 40S ribosomal protein SA (RPSA) was downregulated in parkin virus-infected cells, and cytokeratins 8 and 18 exhibited an acid shift in pI value without a change in molecular weight, suggesting that these proteins became phosphorylated in parkin virus-infected cells. The changes in these three proteins were first observed at 60 h postinfection and were most dramatic at 72 h postinfection. Because upregulation of RPSA and dephosphorylation of cytokeratins 8/18 have been linked with tumor progression, these data suggest that parkin may inhibit cell growth, at least in part, by decreasing RPSA expression and inducing phosphorylation of cytokeratin 8/18.