Identification of distinct slow mode of reversible adaptation of pancreatic ductal adenocarcinoma to the prolonged acidic pH microenvironment.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic neoplasm with high metastatic potential and poor clinical outcome. Like other solid tumors, PDAC in the early stages is often asymptomatic, and grows very slowly under a distinct acidic pHe (extracellular pH) microenvironment. However, most previous studies have only reported the fate of cancerous cells upon cursory exposure to acidic pHe conditions. Little is known about how solid tumors-such as the lethal PDAC originating within the pancreatic duct-acinar system that secretes alkaline fluids-evolve to withstand and adapt to the prolonged acidotic microenvironmental stress. METHODS: Representative PDAC cells were exposed to various biologically relevant periods of extracellular acidity. The time effects of acidic pHe stress were determined with respect to tumor cell proliferation, phenotypic regulation, autophagic control, metabolic plasticity, mitochondrial network dynamics, and metastatic potentials. RESULTS: Unlike previous short-term analyses, we found that the acidosis-mediated autophagy occurred mainly as an early stress response but not for later adaptation to microenvironmental acidification. Rather, PDAC cells use a distinct and lengthy process of reversible adaptive plasticity centered on the early fast and later slow mitochondrial network dynamics and metabolic adjustment. This regulates their acute responses and chronic adaptations to the acidic pHe microenvironment. A more malignant state with increased migratory and invasive potentials in long-term acidosis-adapted PDAC cells was obtained with key regulatory molecules being closely related to overall patient survival. Finally, the identification of 34 acidic pHe-related genes could be potential targets for the development of diagnosis and treatment against PDAC. CONCLUSIONS: Our study offers a novel mechanism of early rapid response and late reversible adaptation of PDAC cells to the stress of extracellular acidosis. The presence of this distinctive yet slow mode of machinery fills an important knowledge gap in how solid tumor cells sense, respond, reprogram, and ultimately adapt to the persistent microenvironmental acidification.

Assessment of Brain Functional Activity Using a Miniaturized Head-Mounted Scanning Photoacoustic Imaging System in Awake and Freely Moving Rats.

Understanding the relationship between brain function and natural behavior remains a significant challenge in neuroscience because there are very few convincing imaging/recording tools available for the evaluation of awake and freely moving animals. Here, we employed a miniaturized head-mounted scanning photoacoustic imaging (hmPAI) system to image real-time cortical dynamics. A compact photoacoustic (PA) probe based on four in-house optical fiber pads and a single custom-made 48-MHz focused ultrasound transducer was designed to enable focused dark-field PA imaging, and miniature linear motors were included to enable two-dimensional (2D) scanning. The total dimensions and weight of the proposed hmPAI system are only approximately 50 × 64 × 48 mm and 58.7 g (excluding cables). Our ex vivo phantom experimental tests revealed that a spatial resolution of approximately 0.225 mm could be achieved at a depth of 9 mm. Our in vivo results further revealed that the diameters of cortical vessels draining into the superior sagittal sinus (SSS) could be clearly imaged and continuously observed in both anesthetized rats and awake, freely moving rats. Statistical analysis showed that the full width at half maximum (FWHM) of the PA A-line signals (relative to the blood vessel diameter) was significantly increased in the selected SSS-drained cortical vessels of awake rats (0.58 ± 0.17 mm) compared with those of anesthetized rats (0.31 ± 0.09 mm) (p < 0.01, paired t-test). In addition, the number of pixels in PA B-scan images (relative to the cerebral blood volume (CBV)) was also significantly increased in the selected SSS-drained blood vessels of awake rats (107.66 ± 23.02 pixels) compared with those of anesthetized rats (81.99 ± 21.52 pixels) (p < 0.01, paired t-test). This outcome may result from a more active brain in awake rats than in anesthetized rats, which caused cerebral blood vessels to transport more blood to meet the increased nutrient demand of the tissue, resulting in an obvious increase in blood vessel volume. This hmPAI system was further validated for utility in the brains of awake and freely moving rats, showing that their natural behavior was unimpaired during vascular imaging, thereby providing novel opportunities for studies of behavior, cognition, and preclinical models of brain diseases.

An Adjustable Dark-Field Acoustic-Resolution Photoacoustic Imaging System with Fiber Bundle-Based Illumination.

Photoacoustic (PA) imaging has become one of the major imaging methods because of its ability to record structural information and its high spatial resolution in biological tissues. Current commercialized PA imaging instruments are limited to varying degrees by their bulky size (i.e., the laser or scanning stage) or their use of complex optical components for light delivery. Here, we present a robust acoustic-resolution PA imaging system that consists of four adjustable optical fibers placed 90° apart around a 50 MHz high-frequency ultrasound (US) transducer. In the compact design concept of the PA probe, the relative illumination parameters (i.e., angles and fiber size) can be adjusted to fit different imaging applications in a single setting. Moreover, this design concept involves a user interface built in MATLAB. We first assessed the performance of our imaging system using in vitro phantom experiments. We further demonstrated the in vivo performance of the developed system in imaging (1) rat ear vasculature, (2) real-time cortical hemodynamic changes in the superior sagittal sinus (SSS) during left-forepaw electrical stimulation, and (3) real-time cerebral indocyanine green (ICG) dynamics in rats. Collectively, this alignment-free design concept of a compact PA probe without bulky optical lens systems is intended to satisfy the diverse needs in preclinical PA imaging studies.

In Vivo Assessment of Hypoxia Levels in Pancreatic Tumors Using a Dual-Modality Ultrasound/Photoacoustic Imaging System.

Noninvasive anatomical and functional imaging has become an essential tool to evaluate tissue oxygen saturation dynamics in preclinical or clinical studies of hypoxia. Our dual-wavelength technique for photoacoustic (PA) imaging based on the differential absorbance spectrum of oxyhemoglobin (oxy-Hb) and deoxyhemoglobin (deoxy-Hb) can quantify tissue oxygen saturation using the intrinsic contrast property. PA imaging of tissue oxygen saturation can be used to monitor tumor-related hypoxia, which is a particularly relevant functional parameter of the tumor microenvironment that has a strong influence on tumor aggressiveness. The simultaneous acquisition of anatomical and functional information using dual-modality ultrasound (US) and PA imaging technology enhances the preclinical applicability of the method. Here, the developed dual-modality US/PA system was used to measure relative tissue oxygenation using the dual-wavelength technique. Tissue oxygen saturation was quantified in a pancreatic tumor mouse model. The differences in tissue oxygenation were detected by comparing pancreatic samples from normal and tumor-bearing mice at various time points after implantation. The use of an in vivo pancreatic tumor model revealed changes in hypoxia at various stages of tumor growth. The US/PA imaging data positively correlated with the results of immunohistochemical staining for hypoxia. Thus, our dual-modality US/PA imaging system can be used to reliably assess and monitor hypoxia in pancreatic tumor mouse models. These findings enable the use of a combination of US and PA imaging to acquire anatomical and functional information on tumor growth and to evaluate treatment responses in longitudinal preclinical studies.

The ligand-mediated affinity of brain-type fatty acid-binding protein for membranes determines the directionality of lipophilic cargo transport.

The intracellular transport of lipophilic cargoes is a highly dynamic process. In eukaryotic cells, the uptake and release of long-chain fatty acids (LCFAs) are executed by fatty-acid binding proteins. However, how these carriers control the directionality of cargo trafficking remains unclear. Here, we revealed that the unliganded archetypal Drosophila brain-type fatty acid-binding protein (dFABP) possesses a stronger binding affinity than its liganded counterpart for empty nanodiscs (ND). Titrating unliganded dFABP and nanodiscs with LCFAs rescued the broadening of FABP cross-peak intensities in HSQC spectra from a weakened protein-membrane interaction. Two out of the 3 strongest LCFA contacting residues in dFABP identified by NMR HSQC chemical shift perturbation (CSP) are also part of the 30 ND-contacting residues (out of the total 130 residues in dFABP), revealed by attenuated TROSY signal in the presence of lipid ND to apo-like dFABP. Our crystallographic temperature factor data suggest enhanced αII helix dynamics upon LCFA binding, compensating for the entropic loss in the ßC-D/ßE-F loops. The aliphatic tail of bound LCFA impedes the charge-charge interaction between dFABP and the head groups of the membrane, and dFABP is prone to dissociate from the membrane upon ligand binding. We therefore conclude that lipophilic ligands participate directly in the control of the functionally required membrane association and dissociation of FABPs.

Lysosomal cysteine protease cathepsin S is involved in cancer cell motility by regulating store-operated Ca2+ entry.

Cathepsin S (CTSS), a lysosomal cysteine protease, has been reported to be associated with extracellular matrix (ECM) degradation, thus promoting cell migration and invasion, but whether CTSS regulates other intracellular mechanisms during metastasis remains unknown. The expression of CTSS was knocked down using siRNA transfection, and enzymatic activity was inhibited by the highly-selective CTSS inhibitor RJW-58. The results of in vitro functional assays, western blot analysis, and an in vivo colonization model demonstrated that CTSS was positively related to cellular adhesive ability. Moreover, both CTSS knockdown and inhibition significantly decreased Ca2+ influx via store-operated Ca2+ entry (SOCE) without changing STIM1 and Orai1 expression levels, while RJW-58 dose-dependently reduced the activation of the Ca2+-dependent downstream effectors, NFAT1 and Rac1. The results of immunoprecipitation assays demonstrated that CTSS could bind to STIM1, which was reversed by CTSS inhibition. In addition, confocal microscopy and super-resolution imaging showed that CTSS inhibition led to STIM1 puncta accumulation in the endoplasmic reticulum and reduced the interaction between active STIM1 and EB1. In conclusion, we have demonstrated for the first time that the lysosomal cysteine protease, CTSS, plays an important role in mediating Ca2+ homeostasis by regulating STIM1 trafficking, which leads to the suppression of cell migration and invasion.

Preventive hypothermia as a neuroprotective strategy for paclitaxel-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe adverse effect that occurs secondary to anticancer treatments and has no known preventive or therapeutic strategy. Therapeutic hypothermia has been shown to be effective in protecting against central and peripheral nervous system injuries. However, the effects of therapeutic hypothermia on CIPN have rarely been explored. We induced lower back hypothermia (LBH) in an established paclitaxel-induced CIPN rat model and found that the paclitaxel-induced impairments observed in behavioral, electrophysiological, and histological impairments were inhibited by LBH when applied at an optimal setting of 24°C to the sciatic nerve and initiated 90 minutes before paclitaxel infusion. Lower back hypothermia also inhibited the paclitaxel-induced activation of astroglia and microglia in the spinal cord and macrophage infiltration into and neuronal injury in the dorsal root ganglia and sciatic nerves. Furthermore, LBH decreased the local blood flow and local tissue concentrations of paclitaxel. Finally, in NOD/SCID mice inoculated with cancer cells, the antiproliferative effect of paclitaxel was not affected by the distal application of LBH. In conclusion, our findings indicate that early exposure to regional hypothermia alleviates paclitaxel-induced peripheral neuropathy. Therapeutic hypothermia may therefore represent an economical and nonpharmaceutical preventive strategy for CIPN in patients with localized solid tumors.

A clamp-like orientation of basic residues set in a parallelogram is essential for heparin binding.

While the majority of studies have focused on the biological roles of heparin-binding proteins, relatively little is known about their key residues and structural elements responsible for heparin interaction. In this study, we employed the IgG-binding domain B1 of Streptococcal protein G as a miniature scaffold to investigate how certain positively charged residues within the ß-sheet conformation become favorable for heparin binding. By performing a series of arginine substitution mutations followed by gain-of-heparin-binding analysis, we deduced that a clamp-like orientation with discontinuous basic residues separated by ~ 5 Å with ~ 100° interior angle is advantageous for high heparin affinity.

AKT3 promotes prostate cancer proliferation cells through regulation of Akt, B-Raf, and TSC1/TSC2.

The qRT-PCR analysis of 139 clinical samples and analysis of 150 on-line database clinical samples indicated that AKT3 mRNA expression level was elevated in primary prostate tumors. Immunohistochemical staining of 65 clinical samples revealed that AKT3 protein expression was higher in prostate tumors of stage I, II, III as compared to nearby normal tissues. Plasmid overexpression of AKT3 promoted cell proliferation of LNCaP, PC-3, DU-145, and CA-HPV-10 human prostate cancer (PCa) cells, while knockdown of AKT3 by siRNA reduced cell proliferation. Overexpression of AKT3 increased the protein expression of total AKT, phospho-AKT S473, phospho-AKT T308, B-Raf, c-Myc, Skp2, cyclin E, GSK3ß, phospho-GSK3ß S9, phospho-mTOR S2448, and phospho-p70S6K T421/S424, but decreased TSC1 (tuberous sclerosis 1) and TSC2 (tuberous Sclerosis Complex 2) proteins in PC-3 PCa cells. Overexpression of AKT3 also increased protein abundance of phospho-AKT S473, phospho-AKT T308, and B-Raf but decreased expression of TSC1 and TSC2 proteins in LNCaP, DU-145, and CA-HPV-10 PCa cells. Oncomine datasets analysis suggested that AKT3 mRNA level was positively correlated to BRAF. Knockdown of AKT3 in DU-145 cells with siRNA increased the sensitivity of DU-145 cells to B-Raf inhibitor treatment. Knockdown of TSC1 or TSC2 promoted the proliferation of PCa cells. Our observations implied that AKT3 may be a potential therapeutic target for PCa treatment.

Caffeic Acid phenethyl ester is a potential therapeutic agent for oral cancer.

Head and neck cancers, which affect 650,000 people and cause 350,000 deaths per year, is the sixth leading cancer by cancer incidence and eighth by cancer-related death worldwide. Oral cancer is the most common type of head and neck cancer. More than 90% of oral cancers are oral and oropharyngeal squamous cell carcinoma (OSCC). The overall five-year survival rate of OSCC patients is approximately 63%, which is due to the low response rate to current therapeutic drugs. In this review we discuss the possibility of using caffeic acid phenethyl ester (CAPE) as an alternative treatment for oral cancer. CAPE is a strong antioxidant extracted from honeybee hive propolis. Recent studies indicate that CAPE treatment can effectively suppress the proliferation, survival, and metastasis of oral cancer cells. CAPE treatment inhibits Akt signaling, cell cycle regulatory proteins, NF-κB function, as well as activity of matrix metalloproteinase (MMPs), epidermal growth factor receptor (EGFR), and Cyclooxygenase-2 (COX-2). Therefore, CAPE treatment induces cell cycle arrest and apoptosis in oral cancer cells. According to the evidence that aberrations in the EGFR/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling, NF-κB function, COX-2 activity, and MMPs activity are frequently found in oral cancers, and that the phosphorylation of Akt, EGFR, and COX-2 correlates to oral cancer patient survival and clinical progression, we believe that CAPE treatment will be useful for treatment of advanced oral cancer patients.

Ethanol extracts of fruiting bodies of Antrodia cinnamomea suppress CL1-5 human lung adenocarcinoma cells migration by inhibiting matrix metalloproteinase-2/9 through ERK, JNK, p38, and PI3K/Akt signaling pathways.

Metastatic cancer attributes to a major cause of cancer death. In this pioneer study, we aimed to investigate how Antrodia cinnamomea (A. cinnamomea), indigenous to Taiwan, affects migration ability of highly metastatic human adenocarcinoma lung cancer cells CL1-5. Our result demonstrated that noncytotoxic ethanol extract of fruiting bodies of A. cinnamomea (EEAC) exhibited a dose-dependent inhibitory effect on motility and migration of the highly metastatic CL1-5 cells. Results of a gelatin zymography assay illustrated that A. cinnamomea repressed the activities of matrix metalloproteinase- (MMP-) 2 and 9 in a dose-dependent manner. A. cinnamomea administration decreased MMP-9 and MMP-2 protein expressions from Western blotting assay, whereas the expression of the tissue inhibitors of MMP (TIMP-1 and TIMP-2) increased. Additional study disclosed that A. cinnamomea suppressed FAK, ERK1/2, p38, AKT, and JNK1/2 phosphorylation, and also PI3K and Rac-1 were found decreased. Further, treatment of CL1-5 cells with inhibitors specific for PI3K (LY294002), ERK1/2 (PD98059), JNK (SP600125), and p38 MAPK (SB203580) decreased the expression of MMP-2 and MMP-9. Taken together, EEAC induced FAK phosphorylation and exhibited its antimigration activities via the PI3K/AKT and MAPK signalings in CL1-5 cells. This is the pioneer study verifying the antimigration activity of A. cinnamomea against human lung adenocarcinoma CL1-5 cancer cells [corrected].

Suppression of the invasion and migration of cancer cells by SERPINB family genes and their derived peptides.

Apart from SERPINB2 and SERPINB5, the roles of the remaining 13 members of the human SERPINB family in cancer metastasis are still unknown. In the present study, we demonstrated that most of these genes are differentially expressed in tumor tissues compared to matched normal tissues from lung or breast cancer patients. Overexpression of each SERPINB gene effectively suppressed the invasiveness and motility of malignant cancer cells. Among all of the genes, the SERPINB1, SERPINB5 and SERPINB7 genes were more potent, and the inhibitory effect was further enhanced by co-expression of any two of them. In addition, single treatment of the synthetic peptides corresponding to the P5-P5' sequences of the reactive center loop (RCL) of SERPINB1, SERPINB5 or SERPINB7 markedly suppressed the invasive and migratory properties of the cancer cells in a dose-dependent manner. More significantly, combination treatment of these peptides in cancer cells further improved the suppressive effect by 20-40%. Here, we determined the expression of all SERPINB family members in lung and breast cancer patients, and identified those members with potent inhibitory ability toward invasion and migration, and designed RCL-derived peptides to suppress the malignancy of cancer cells. Forced re-expression of these anti-invasive SERPINB genes or application of the SERPINB RCL-peptides may provide a reasonable strategy against lethal cancer metastasis.

Targeting cathepsin S induces tumor cell autophagy via the EGFR-ERK signaling pathway.

Cathepsin S is a cellular cysteine protease, which is frequently over-expressed in human cancer cells and plays important role in tumor metastasis. However, the role of cathepsin S in regulating cancer cell survival and death remains undefined. The aim of this study was to determine whether targeting cathepsin S could induce autophagy/apoptosis in cancer cells. In this study, we demonstrated that targeting cathepsin S by either specific small molecular inhibitors or cathepsin S siRNA induced autophagy and subsequent apoptosis in human cancer cells, and the induction of autophagy was dependent on the phosphorylation of EGFR and activation of the EGFR-related ERK/MAPK-signaling pathway. In conclusion, the current study reveals that cathepsin S plays an important role in the regulation of cell autophagy through interference with the EGFR-ERK/MAPK-signaling pathway.

Epigenetic activation of human kallikrein 13 enhances malignancy of lung adenocarcinoma by promoting N-cadherin expression and laminin degradation.

The tissue kallikrein (KLK) family contains 15 genes (KLK1-KLK15) tandemly arranged on chromosome 19q13.4 that forms the largest cluster of contiguous protease genes in the human genome. Here, we provide mechanistic evidence showing that the expression of KLK13, one of the most recently identified family members, is significantly up-regulated in metastatic lung adenocarcinoma. Whilst overexpression of KLK13 resulted in an increase in malignant cell behavior, knockdown of its endogenous gene expression caused a significant decrease in cell migratory and invasive properties. Functional studies further demonstrated that KLK13 is activated via demethylation of its upstream region. The elevated KLK13 protein then enhances the ability of tumor cells to degrade extracellular laminin that, subsequently, facilitates cell metastatic potential in the in vivo SCID mouse xenograft model. KLK13 was also found to induce the expression of N-cadherin to help promote tumor cell motility. Together, these results reveal the enhancing effects of KLK13 on tumor cell invasion and migration, and that it may serve as a diagnostic/prognostic marker and a potential therapeutic target for lung cancer.

Mitochondrial proteomics analysis of tumorigenic and metastatic breast cancer markers.

Mitochondria are key organelles in mammary cells responsible for several cellular functions including growth, division, and energy metabolism. In this study, mitochondrial proteins were enriched for proteomics analysis with the state-of-the-art two-dimensional differential gel electrophoresis and matrix-assistant laser desorption ionization-time-of-flight mass spectrometry strategy to compare and identify the mitochondrial protein profiling changes between three breast cell lines with different tumorigenicity and metastasis. The proteomics results demonstrate more than 1,500 protein features were resolved from the equal amount pooled from three purified mitochondrial proteins, and 125 differentially expressed spots were identified by their peptide finger print, in which, 33 identified proteins belonged to mitochondrial proteins. Eighteen out of these 33 identified mitochondrial proteins such as SCaMC-1 have not been reported in breast cancer research to our knowledge. Additionally, mitochondrial protein prohibitin has shown to be differentially distributed in mitochondria and in nucleus for normal breast cells and breast cancer cell lines, respectively. To sum up, our approach to identify the mitochondrial proteins in various stages of breast cancer progression and the identified proteins may be further evaluated as potential breast cancer markers in prognosis and therapy.

Design and synthesis of alpha-ketoamides as cathepsin S inhibitors with potential applications against tumor invasion and angiogenesis.

A series of small molecules bearing an alpha-ketoamide warhead were synthesized and evaluated for their ability to inhibit cathepsin S, a key proteolytic enzyme upregulated in many cancers during tumor progression and metastasis. Most of the synthetic compounds were noncytotoxic, but several robustly inhibited cathepsin S (IC(50) < 10 nM) and potently suppressed cell migration, invasion, and capillary tube formation. These results highlight the potential of alpha-ketoamide therapy for preventing or delaying cancer spread.

Molecular characterization of invasive subpopulations from an esophageal squamous cell carcinoma cell line.

BACKGROUND: Once diagnosed, esophageal cancer has a very low overall 5-year survival rate. This study investigates the mechanisms behind the invasiveness and severity of esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: Transwell invasion chamber was used to subdivide one Taiwanese ESCC cell line, CE81T/VGH, into sublines (CE81T-0, CE81T-1, CE81T-2, CE81T-3, and CE81T-4) in four rounds of assays; the most invasive were identified, and various factors related to their invasiveness measured. RESULTS: CE81T-1, CE81T-2, CE81T-3 and CE81T-4 sublines were significantly more invasive than the parental cells (CE81T/VGH) and CE81T-0 subline. CE81T-1 and CE81T-4, the sublines we chose to study further, had significantly greater colony-forming ability (3.5- to 2.7-fold) and wound migrating activity (1.95- to 2.6-fold) than the parental cells in vitro (p<0.01). They also displayed greater tumorigenesis in immunodeficient BALB/c Foxlnn mice than the parental cells. We found an inverse correlation between expression of tissue inhibitor of metalloproteinase-2 and invasive ability, and a significant positive correlation between expressions of matrix metalloproteinase-1, vimentin, and p-Src (pY416) in these cell lines and their invasiveness (all p<0.05). CONCLUSION: The subline model may be used to study the molecular and genetic mechanisms underlying the invasion and metastasis of ESCC.

Cooperation between NRF-2 and YY-1 transcription factors is essential for triggering the expression of the PREPL-C2ORF34 bidirectional gene pair.

BACKGROUND: Many mammalian genes are organized as bidirectional (head-to-head) gene pairs with the two genes separated only by less than 1 kb. The transcriptional regulation of these bidirectional gene pairs remains largely unclear, but a few studies have suggested that the two closely adjacent genes in divergent orientation can be co-regulated by a single transcription factor binding to a specific regulatory fragment. Here we report an evolutionarily conserved bidirectional gene pair, known as the PREPL-C2ORF34 gene pair, whose transcription relies on the synergic cooperation of two transcription factors binding to an intergenic bidirectional minimal promoter. RESULTS: While PREPL is present primarily in brain and heart, C2ORF34 is ubiquitously and abundantly expressed in almost all tissues. Genomic analyses revealed that these two non-homologous genes are adjacent in a head-to-head configuration on human chromosome 2p21 and separated by only 405 bp. Within this short intergenic region, a 243-bp GC-rich segment was demonstrated to function as a bidirectional minimal promoter to initiate the transcription of both flanking genes. Two key transcription factors, NRF-2 and YY-1, were further identified to coordinately participate in driving both gene expressions in an additive manner. The functional cooperation between these two transcription factors, along with their genomic binding sites and some cis-acting repressive elements, are essential for the transcriptional activation and tissue distribution of the PREPL-C2ORF34 bidirectional gene pair. CONCLUSION: This study provides new insights into the complex transcriptional mechanism of a mammalian head-to-head gene pair which requires cooperative binding of multiple transcription factors to a bidirectional minimal promoter of the shared intergenic region.

Identification of trypsin-inhibitory site and structure determination of human SPINK2 serine proteinase inhibitor.

Human serine proteinase inhibitor Kazal-type 2 (SPINK2) functions as a trypsin/acrosin inhibitor and is synthesized mainly in the testis and seminal vesicle where its activity is engaged in fertility. The SPINK2 protein contains a typical Kazal domain composed by six cysteine residues forming three disulfide bridges. The expression of SPINK2 is closely related to cancer such as lymphomas, in that a high transcript level of SPINK2 in patients with primary cutaneous follicle center cell lymphomas have better prognosis with lower mortality. To clarify the role of SPINK2 in cancer, we performed quantitative real-time PCR and showed that the expression level of SPINK2 is significantly elevated in most leukemia cell lines except B-lymphoblast TK-6 cells. The molecular function and structural features of SPINK2 were also investigated by employing the recombinant active and mutant inactive SPINK2 proteins to determine its key P2-P2' (Pro(23)-Arg(24)-His(25)-Phe(26)) active site. The inhibition assay results demonstrated that Arg(24) at the P1 site is crucial for the specificity of SPINK2 on target enzyme. Although His(25) at the P1' and Phe(26) at the P2' residues are also involved in trypsin-SPINK2 interaction, Pro(23) at the P2 site may not be directly participated in interacting with trypsin. In addition, we determined the 3D solution structure of SPINK2 and used this structure to predict the SPINK2-proteinase complex structure and binding properties. These studies not only provide critical information about the structural properties and biophysical features of the SPINK2 proteinase inhibitor, but also suggest its important role in tumor progression and response to treatment.

ALA-PDT results in phenotypic changes and decreased cellular invasion in surviving cancer cells.

BACKGROUND AND OBJECTIVES: The mechanisms of photodynamic therapy (PDT) have been studied on the cellular and tissue levels. However, the cellular behaviors of cancer cells survived from PDT are still not clear. This study attempted to investigate the influence of 5-aminolevulinic acid (ALA)-based PDT on the invasion ability as well as molecular changes in surviving cancer cells and their progeny. MATERIALS AND METHODS: The systematic effects of ALA-PDT were evaluated using human invasive carcinoma cells (lung adenocarcinoma CL1-5 cells, melanoma A375 cells and breast carcinoma MDA-MB-231 cells). To study the cellular behaviors of surviving cancer cells, PDT-derived variants were established as stable cell lines after consecutive treatment with ALA-PDT. Scratch wound assay and invasion assay were performed to evaluate the migration and invasion ability in the surviving cancer cells and the established PDT-derived variants. RT-PCR and immunoblot analysis were performed to examine the expression levels of epidermal growth factor receptor (EGFR). RESULTS: Though ALA-PDT caused differential phototoxicity among these invasive carcinoma cells, reduced migration was found in all the surviving cancer cells Compared to parental cancer cells, the established PDT-derived variants exerted significant phenotypic changes of cellular morphology, reduced mitochondrial function and a suppressed cellular invasiveness. Furthermore, correlated with the reduced invasion ability, expression of EGFR was downregulated in these established PDT-derived variants. CONCLUSIONS: Except for direct cell killing, ALA-PDT could reduce EGFR expression and invasion ability of the surviving cancer cells and these effects could further pass to the progeny. The results from this study provide insights into a new mechanism by which PDT might affect cellular behaviors and tumor metastasis.