Polypharmacological Drug Design Guided by Integrating Phenotypic and Restricted Fragment Docking Strategies.

Polypharmacological drugs are of great value for treating complex human diseases by the combinative modulation of several biological targets. However, multitarget drug design with more than two targets is challenging and generally discovered by serendipity. Herein, a restricted fragment docking (RFD) computational method combined with a phenotypic discovery approach was developed for rational polypharmacological drug design. Via genetic and drug combination studies in a microglial phenotype, we first identified novel synergistic effects by triple target modulation toward RIPK1, MAP4K4, and ALK. Drawing on the RFD method to explore virtual chemical spaces in three target pockets, we identified a lead compound, LP-10d, that precisely modulated RIPK1/MAP4K4/ALK for synergistic microglial protection with low nanomolar potency. LP-10d showed polypharmacology against multiple neuropathologies in the 3xTg Alzheimer's disease mouse model. Our study revealed a potential application of the RFD method, which is valuable to further polypharmacological drug discovery involved in clinical studies for treating complex human diseases.

Discovery of Covalent MLKL PROTAC Degraders via Optimization of a Theophylline Derivative Ligand for Treating Necroptosis.

Mixed lineage kinase domain-like pseudokinase (MLKL) initiates necroptosis and could serve as a therapeutic target related to a series of human diseases. Proteolysis-targeting chimeras (PROTACs) are useful tools for degrading pathological proteins and blocking disease processes. Using computer-aided modeling and molecular dynamics simulations, we developed a series of covalent MLKL PROTACs by linking and optimizing a theophylline derivative that covalently targets MLKL. Via structure-activity relationship studies, MP-11 was identified as a potent MLKL PROTAC degrader. Furthermore, MP-11 showed lower toxicity than the original MLKL ligand, exhibiting nanomolar-scale antinecroptotic activity on human cell lines. Xenograft model studies showed that MP-11 effectively degraded MLKL in vivo. Importantly, our study demonstrates that the covalent binding strategy is an effective approach for designing MLKL-targeting PROTACs, serving as a model for developing PROTACs to treat future necroptosis-related human diseases.

Improving tumor sensitivity by the introduction of an ester chain to triaryl derivatives targeting PD-1/PD-L1.

PD-1/PD-L1 pathway blockade is a promising immunotherapy for the treatment of cancer. In this manuscript, a series of triaryl compounds containing ester chains were designed and synthesized based on the pharmacophore studies of the lead BMS-1. After several SAR iterations, 22 showed the best biochemical activity binding to hPD-L1 with an IC50 of 1.21 nM in HTRF assay, and a KD value of 5.068 nM in SPR analysis. Cell-based experiments showed that 22 effectively promoted A549 cell death by restoring T-cell immune function. 22 showed significant in vivo antitumor activity in a 4T1 mouse model without obvious toxicity, with a TGI rate of 67.8 % (20 mg/kg, ip). Immunohistochemistry data indicated that 22 activates the immune activity in tumors. These results suggest that 22 is a promising compound for further development of PD-1/PD-L1 inhibitor for cancer therapy.

Progress in approved drugs from natural product resources.

Natural products (NPs) have consistently played a pivotal role in pharmaceutical research, exerting profound impacts on the treatment of human diseases. A significant proportion of approved molecular entity drugs are either directly derived from NPs or indirectly through modifications of NPs. This review presents an overview of NP drugs recently approved in China, the United States, and other countries, spanning various disease categories, including cancers, cardiovascular and cerebrovascular diseases, central nervous system disorders, and infectious diseases. The article provides a succinct introduction to the origin, activity, development process, approval details, and mechanism of action of these NP drugs.

Dual target PARP1/EZH2 inhibitors inducing excessive autophagy and producing synthetic lethality for triple-negative breast cancer therapy.

Currently available PARP inhibitors are mainly used for the treatment of BRCA-mutated triple-negative breast cancer (TNBC), with a narrow application range of approximately 15% of patients. Recent studies have shown that EZH2 inhibitors have an obvious effect on breast cancer xenograft models and can promote the sensitivity of ovarian cancer cells to PARP inhibitors. Here, a series of new dual-target PARP1/EZH2 inhibitors for wild-BRCA type TNBC were designed and synthesized. SAR studies helped us identify compound 12e, encoded KWLX-12e, with good inhibitory activity against PARP1 (IC50 = 6.89 nM) and EZH2 (IC50 = 27.34 nM). Meanwhile, KWLX-12e showed an optimal cytotoxicity against MDA-MB-231 cells (IC50 = 2.84 µM) and BT-549 cells (IC50 = 0.91 µM), with no toxicity on normal breast cell lines. KWLX-12e also exhibited good antitumor activity with the TGI value of 75.94%, more effective than Niraparib plus GSK126 (TGI = 57.24%). Mechanistic studies showed that KWLX-12e achieved synthetic lethality indirectly by inhibiting EZH2 to increase the sensitivity to PARP1, and induced cell death by regulating excessive autophagy. KWLX-12e is expected to be a potential candidate for the treatment of TNBC.

Indirubin derivatives as bifunctional molecules inducing DNA damage and targeting PARP for the treatment of cancer.

Based on the facts that significant synergistic effect existed between PARP inhibitors and DNA damage agents and the DNA damage caused by indirubin's derivatives, we herein adopted the strategy to combine the pharmacophores of PARP inhibitors and the unique scaffold of indirubin to design a series of bifunctional molecules inducing DNA damage and targeting PARP. After SAR studies, the most potent compound 12a, encoded as KWWS-12a, exhibited improved inhibitory effect against PARP1 compared with PARP1 inhibitor Olaparib (IC50 = 1.89 nM vs 7.48 nM) and enhanced antiproliferative activities than the combination of Olaparib and indirubin-3'-monoxime towards HCT-116 cells (IC50 = 0.31 µM vs 1.37 µM). In the normal NCM-460 cells, 12a showed low toxicity (IC50 > 60 µM). The mechanism research indicated that 12a could increase the levels of γH2AX concentration dependently, arrest the cell cycle in S phase and induce apoptosis in HCT-116 cells. In vivo experiments showed that 12a displayed more significant antitumor potential than that of the positive controls. Our studies demonstrated that 12a could be a promising candidate for cancer therapy.

Design, synthesis and mechanism studies of dual EZH2/BRD4 inhibitors for cancer therapy.

Aberrant expression of EZH2 is frequently observed in cancers, and the EZH2 inhibitors are only effective in hematological malignancies and almost noneffective against solid tumors. It has been reported that the combination of EZH2 and BRD4 inhibitors may be a promising strategy to treat solid tumors being insensitive to EZH2 inhibitors. Thus, a series of EZH2/BRD4 dual inhibitors were designed and synthesized. The optimized compound 28, encoded as KWCX-28, was the most potential compound by the SAR studies. Further mechanism studies showed that KWCX-28 inhibited HCT-116 cells proliferation (IC50 = 1.86 µM), induced HCT-116 cells apoptosis, arrested cell cycle arrest at G0/G1 phase and resisted the histone 3 lysine 27 acetylation (H3K27ac) upregulation. Therefore, KWCX-28 was a potential dual EZH2/BRD4 inhibitors for treating solid tumors.

Histone Deacetylase and Enhancer of Zeste Homologue 2 Dual Inhibitors Presenting a Synergistic Effect for the Treatment of Hematological Malignancies.

Aberrance of epigenetic modification is one of the important factors leading to hematological malignancies. Histone deacetylase (HDAC) inhibitors and enhancers of zeste homologue 2 (EZH2) inhibitors are demonstrated to be significant epigenic modulators. Cocktail therapy of HDAC inhibitors and EZH2 inhibitors was demonstrated to be a promising strategy in hematological malignancies. We designed HDAC and EZH2 dual inhibitors based on the strong synergistic effect of SAHA and GSK126. Compound 20 exhibited excellent inhibitory activity against HDAC1 (IC50 = 0.12 µM) and EZH2 (IC50 = 0.059 µM), it also showed good antiproliferation activity against MV4-11 (IC50 = 0.17 µM), which has more potential than the cocktail therapy of SAHA and GSK126 (IC50 = 0.40 µM). 20 suppressed tumor growth in vivo, which was as good as the combination therapy. These results suggested that 20 may be a promising drug candidate for treating hematological malignancies.

Discovery of precision targeting EZH2 degraders for triple-negative breast cancer.

EZH2 is usually overexpressed in TNBC and other tumors, which has a great influence on the occurrence, development and prognosis of tumors. However, current EZH2 inhibitors, including Tazemetostat and GSK126, affect the methyl catalytic capacity of EZH2 and have little effect on the tumorigenic activity of EZH2 itself, resulting in poor efficacy against most solid tumors. Herein, we designed and optimized proteolytic targeting chimeras (PROTACs) precision targeting EZH2. The most active PROTAC molecule U3i has a high affinity for PRC2 complex (KD = 16.19 nM) and show good inhibitory effects on MDA-MB-231 (IC50 = 0.57 µM) and MDA-MB-468 (IC50 = 0.38 µM) cells. Compared with that of the GSK126, the growth inhibitory activities of U3i against these two TNBC cells increased by approximately 20- and 30-fold. Further studies showed that U3i can degrade PRC2 complex in TNBC cells, induce apoptosis, and cause little damage to normal cells. Therefore, U3i is a potential anticancer molecule for TNBC treatment.

Harmine-based dual inhibitors targeting histone deacetylase (HDAC) and DNA as a promising strategy for cancer therapy.

Overexpression of histone deacetylases (HDACs) are observed in different types of cancers, but histone deacetylase inhibitors (HDACIs) have not shown significant efficacy as monotherapy against solid tumors. Recently, studies demonstrated that it is promising to combine HDACIs with DNA damage agents to improve DNA damage level to gain better effect on treating solid tumor. Harmine has been demonstrated to cause DNA damage by intercalating DNA. Therefore, we designed a series of harmine-based inhibitors targeting HDAC and DNA with multi-target strategy, the most potential compound 27 could bind to DNA and cause DNA damage. Furthermore 27 caused cells apoptosis through p53 signaling pathway, and exhibited significant anti-proliferation effects against HCT-116 cells (IC50 = 1.41 µM). As a DNA damage agent, 27 displayed low toxicity in normal cells. Compound 27 was demonstrated as a dual inhibitor targeting HDAC (HDAC1 IC50 = 0.022 µM and HDAC6 IC50 = 0.45 µM) and DNA, and had the potential in the treatment of solid tumor.

Parthenolide Derivatives as PKM2 Activators Showing Potential in Colorectal Cancer.

As a vital kinase in the glycolysis system, PKM2 is extensively expressed in colorectal cancer (CRC) to support the energy and biosynthetic needs. In this study, we designed a series of parthenolide (PTL) derivatives through a stepwise structure optimization, and an excellent derivate 29e showed good activity on PKM2 (AC50 = 86.29 nM) and displayed significant antiproliferative activity against HT29 (IC50 = 0.66 µM) and SW480 (IC50 = 0.22 µM) cells. 29e decreased the expression of total PKM2, prevented nucleus translocation of PKM2 dimer, and inhibited PKM2/STAT3 signaling pathway. 29e remarkably increased OCR and decreased the extracellular acidification rate (ECAR). The antiproliferative effect of 29e depended on PKM2, and the Cys424 of PKM2 was the key binding site. Furthermore, 29e significantly suppressed tumor growth in the HT29 xenograft model without obvious toxicity. These outcomes demonstrate that 29e is a promising drug candidate for the treatment of CRC.

Paeoniae radix alba polysaccharides obtained via optimized extraction treat experimental autoimmune hepatitis effectively.

The extraction process of Paeoniae radix alba polysaccharides (PRAP) was optimized as the liquid-solid ratio of 10.65 mL/g, the extraction time of 2.10 h, and the 2 extraction repetitions through a response surface methodology. The chemical profiles of the obtained PRAP were characterized by measuring the contents of total carbohydrates, total phenolics, uronic acid and protein, and by analyzing the FT-IR spectrum and monosaccharide composition. To determine the therapeutic effects of PRAP on experimental autoimmune hepatitis (EAH), we established an EAH mice model. After treated with PRAP, liver and spleen injuries were reduced, and hepatocyte regeneration and liver function were improved. Further study of the mechanism by which PRAP treats EAH showed that PRAP significantly inhibited oxidative stress in the livers of EAH model mice. More importantly, PRAP inhibited immune inflammatory reactions in EAH model mice, including the hepatic infiltration of inflammatory CD4+ and CD8+ T cells, as well as overexpression of inflammatory cytokines IL-2, IL-6 and IL-10, via inhibition of the NF-κB signaling pathway.

Glucose Oxidase- and UVA-Induced Changes in the Expression Patterns of Seven De-sumoylation Enzymes (SENPs) Are Associated with Cataract Development.

OBJECTIVE: It has been well established that sumoylation acts as an important regulatory mechanism that controls many different cellular processes. We and others have shown that sumoylation plays an indispensable role during mouse eye development. Whether sumoylation is implicated in ocular pathogenesis remains to be further studied. In the present study, we have examined the expression patterns of the de-sumoylation enzymes (SENPs) in the in vitro cataract models induced by glucose oxidase and UVA irradiation. METHODS: Four-week-old C57BL/6J mice were used in our experiments. Lenses were carefully dissected out from mouse eyes and cultured in M199 medium for 12 hours. Transparent lenses (without surgical damage) were selected for experimentation. The lenses were exposed to UVA for 60 min or treated with 20 mU/mL glucose oxidase (GO) to induce cataract formation. The mRNA levels were analyzed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. RESULTS: GO treatment and UVA irradiation can induce cataract formation in lens cultured in vitro. GO treatment significantly down-regulated the mRNA levels for SENPs from 50% to 85%; on the other hand, expression of seven SENP proteins under GO treatment appeared in 3 situations: upregulation for SENP1, 2 and 6; downregulation for SENP 5 and 8; and unchanged for SENP3 and 7. UVA irradiation upregulates the mRNAs for all seven SENPs; In contrast to the mRNA levels for 7 SENPs, the expression levels for 6 SENPs (SENP1-3, 5-6 and 8) appeared down-regulated from 10% to 50%, and only SENP7 was slightly upregulated. CONCLUSION: Our results for the first time established the differentiation expression patterns of 7 de-sumoylation enzymes (SENPs) under treatment by GO or UVA, which provide preliminary data to link sumoylation to stress-induced cataractogenesis.

Heterochromatin protects retinal pigment epithelium cells from oxidative damage by silencing p53 target genes.

Oxidative stress (OS)-induced retinal pigment epithelium (RPE) cell apoptosis is critically implicated in the pathogenesis of age-related macular degeneration (AMD), a leading cause of blindness in the elderly. Heterochromatin, a compact and transcriptional inert chromatin structure, has been recently shown to be dynamically regulated in response to stress stimuli. The functional mechanism of heterochromatin on OS exposure is unclear, however. Here we show that OS increases heterochromatin formation both in vivo and in vitro, which is essential for protecting RPE cells from oxidative damage. Mechanistically, OS-induced heterochromatin selectively accumulates at p53-regulated proapoptotic target promoters and inhibits their transcription. Furthermore, OS-induced desumoylation of p53 promotes p53-heterochromatin interaction and regulates p53 promoter selection, resulting in the locus-specific recruitment of heterochromatin and transcription repression. Together, our findings demonstrate a protective function of OS-induced heterochromatin formation in which p53 desumoylation-guided promoter selection and subsequent heterochromatin recruitment play a critical role. We propose that targeting heterochromatin provides a plausible therapeutic strategy for the treatment of AMD.

Sodium Iodate-Induced Mouse Model of Age-Related Macular Degeneration Displayed Altered Expression Patterns of Sumoylation Enzymes E1, E2 and E3.

PURPOSE: Protein sumoylation is a highly dynamic and reversible post-translational modification, involving covalently conjugation of the small ubiquitin-like modifier (SUMO) to the lysine residue of the target protein. Similar to ubiquitination, sumoylation is catalyzed by E1, E2 and several E3 ligases. However, sumoylation usually does not cause protein degradation but alter the target function through diverse mechanisms. Increasing evidences have shown that sumoylation plays pivotal roles in the pathogenesis of human diseases, including neuron degeneration, cancer and heart disease, etc. We and others have shown that sumoylation is critically implicated in mouse eye development. However, the expression of sumoylation machinery has not been characterized in normal and pathogenic retina. Worldwide, age-related macular degeneration (AMD) is the leading cause of irreversible blindness in aged person. In the present study, we investigated the expression of the major sumoylation enzymes in normal mice and sodium iodateinduced AMD mouse model. METHODS: Four-week-old C57BL/6J mice were used in our experiment. A sterile 1% NaIO3 solution was freshly prepared in PBS from solid NaIO3. Experimental mice were injected with 70 mg/kg NaIO3, and similar volumes of PBS as control. Eyes were enucleated and immersion in FAA fixation overnight and processed for eye cross-sections. After fixation, cross sections eyes were dehydrated, embedded in paraffin, and 6 mm transverse sections were cut using the rotary microtome. Then paraffin sections were stained with hematoxylin and eosin (H&E), and mouse retinal thickness was observed to assess the histopathologic changes. RESULTS: Significantly declined RNA levels of E1, E2 and E3 ligase PIAS1 in NaIO3-injected mouse RPE one day-post treatment. Consistently, the protein level of PIAS1 was also decreased at this time point. At the late stage of treatment (three days post-injection), significantly reduced expression of E1 enzyme SAE1/UBA2 was detected in NaIO3-injected mouse retinas. In the contrary, dramatically increased E3 ligase RanBP2 was found in the injected-retinas. CONCLUSION: Together, our results demonstrated for the first time the dynamic expression of sumoylation pathway enzymes during the progression of retina degeneration induced by oxidative stress. Dynamic expression of E1, E2 and E3 enzymes were found during the time course of RPE and retina degeneration, which revealed the potential regulatory roles of sumoylation in AMD pathogenesis.

Differential Expression of Seven De-sumoylation Enzymes (SENPs) in Major Ocular Tissues of Mouse Eye.

PURPOSE: Protein Sumoylation is one of the most important and prevalent posttranscriptional modification. Increasing evidence have shown that the SENPs (sentrin/SUMOspecific proteases) are critical for steady-state levels of SUMO modification of target proteins, and protein de-sumoylation modulates a great diversity of biological processes including transcription, development, differentiation, neuroprotection, as well as pathogenesis. In the vertebrate eye, we and others have previously shown that sumoylation participated in the differentiation of major ocular tissues including retina and lens. However, the biological significance of seven SENP enzymes: SENP1 to 3 and SENP5 to 8 have not be fully investigated in the ocular tissues. METHODS: The 5 major ocular cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS) or rabbit serum (RBS) and 1% Penicillin- Streptomycin. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. RESULTS: At the mRNA level, all SENPs were highly expressed in retina, and much reduced expression patterns in cornea, lens epithelium and lens fiber. At the protein level, SENP1 to -3, and SENP6 were highly abundant in cornea, while SENP5, SENP7 and SENP8 were enriched in retina, and these SENPs were relatively less abundant in lens tissues. CONCLUSION: Our results for the first time established the differentiation expression patterns of the 7 de-sumoylation enzymes (SENPs), which provides a basis for further investigation of protein desumoylation functions in vertebrate eye.

Localization Analysis of Seven De-sumoylation Enzymes (SENPs) in Ocular Cell Lines.

PURPOSE: It is now well established that protein sumoylation acts as an important regulatory mechanism modulating functions over three thousand proteins. In the vision system, protein conjugation with SUMO peptides can regulate differentiation of multiple ocular tissues. Such regulation is often explored through analysis of biochemical and physiological changes with various cell lines in vitro. We have recently analyzed the expression levels of both mRNAs and proteins for seven de-sumoylation enzymes (SENPs) in five major ocular cell lines. In continuing the previous study, here we have determined their cellular localization of the seven de-sumoylation enzymes (SENP1, 2, 3, 5, 6, 7 and 8) in the above 5 major ocular cell lines using immunocytochemistry. METHODS: The 5 major ocular cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS) or rabbit serum (RBS) and 1% Penicillin- Streptomycin. The localization of the 7 major de-sumoylation enzymes (SENPs) in the 5 major ocular cell lines were determined with immunohistochemistry. The images were captured with a Zeiss LSM 880 confocal microscope. RESULTS: 1) The SENP1 was localized in both cytoplasm and nucleus of 3 human ocular cell lines, FHL124, HLE and ARPE-19; In N/N1003A and αTN4-1, SENP 1 was more concentrated in the cytoplasm. SENP1 appears in patches; 2) SENP2 was distributed in both cytoplasm and nucleus of all ocular cell lines in patches. In HLE and ARPE-19 cells, SENP2 level was higher in nucleus than in cytoplasm; 3) SENP3 was almost exclusively concentrated in the nuclei in all ocular cells except for N/N1003A cells. In the later cells, a substantial amount of SENP3 was also detected in the cytoplasm although nuclear SENP3 level was higher than the cytoplasmic SENP3 level. SENP3 appeared in obvious patches in the nuclei; 4) SENP5 was dominantly localized in the cytoplasm (cellular organelles) near nuclear membrane or cytoplasmic membrane ; 5) SENP6 was largely concentrated in the nuclei of all cell lines except for αTN4-1 cells. In the later cells, a substantial amount of SENP6 was also detected in the cytoplasm although nuclear SENP6 level was higher than the cytoplasmic SENP6 level. 6) SENP7 has an opposite localization pattern between human and animal cell lines. In human cell lines, a majority of SENP7 was localized in nuclei whereas in mouse and rabbit lens epithelial cells, most SENP7 was distributed in the cytoplasm. SENP8 was found present in human cell lines. The 3 human ocular cell lines had relatively similar distribution pattern. In FHL124 and ARPE-19 cells, SENP8 was detected only in the cytoplasm, but in HLE cells, patches of SENP8 in small amount was also detected in the nuclei. CONCLUSIONS: Our results for the first time defined the differential distribution patterns of seven desumoylation enzymes (SENPs) in 5 major ocular cell lines. These results help to understand the different functions of various SENPs in maintaining the homeostasis of protein sumoylation patterns during their functioning processes.

Analysis of the Differential Expression Patterns of Sumoylation Enzymes E1, E2 and E3 in Ocular Cell Lines.

PURPOSE: Protein sumoylation is a well established regulatory mechanism to control many cellular processes such as chromatin structure dynamics, transcriptional regulation of gene expression, cell proliferation and differentiation, cell transformation and carcinogenesis, autophagy and senescence. In the vertebrate vision system, we and others have revealed that sumoylation plays important roles in regulating differentiation of several ocular tissues during eye development. To further elucidate the functional mechanisms of sumoylation, in vitro assay systems are needed. Currently, the five major cell lines including αTN4-1, FHL124, HLE, N/N1003A and ARPE-19 have been extensively used to test the biochemical and molecular aspects of normal vision physiology and various disease processes. Thus, we conducted the study on the expression patterns of the three types of sumoylation enzymes, the activating enzymes SAE1 and UBA2, the conjugating enzyme UBC9, and the ligating enzymes such as RanBP2 and PIAS1 in these ocular cell lines. METHODS: The 5 major ocular cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS) or rabbit serum (RBS) and 1% Penicillin- Streptomycin. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J. RESULTS: we have obtained the following results: 1) For the mRNAs encoding E1 SAE1 and UBA2, E2 UBC9 and E3 PIAS1, the highest level of expression was observed in αTN4-1 cells; For the mRNA encoding RanBP2, the highest level of expression was detected in N/N1003A cells; 2) In contrast to the mRNA expression patterns, a similar level of the SAE1 protein was observed in the all five cell lines, and so is true with UBA2 protein in all cells except for N/N1003A where over fourfold of enrichment in UBA2 protein was observed compared with other cell lines; 3) A similar level of UBC9 protein was also detected in all cells except for N/N1003A where more than one-fold of decrease in UBC9 level was found compared with other cell lines; 4) For E3 ligases, we did not identify the regular PIAS1 band in N/N1003A cells, the remaining cells have a level of PIAS1 with difference of less than 0.6-fold; all cells except for FHL124 cells have a similar level of RanBP2, and a 70% drop in RanBP2 was observed in FHL124 cell. CONCLUSIONS: Our determination of the differential expression patterns of the three types of sumoylation enzymes in the 5 ocular cell lines help to understand sumoylation functions in vertebrate eye.

The small heat shock protein αA-crystallin negatively regulates pancreatic tumorigenesis.

Our recent study has shown that αA-crystallin appears to act as a tumor suppressor in pancreas. Here, we analyzed expression patterns of αA-crystallin in the pancreatic tumor tissue and the neighbor normal tissue from 74 pancreatic cancer patients and also pancreatic cancer cell lines. Immunocytochemistry revealed that αA-crystallin was highly expressed in the normal tissue from 56 patients, but barely detectable in the pancreatic tumor tissue. Moreover, a low level of αA-crystallin predicts poor prognosis for patients with pancreatic duct adenocarcinoma (PDAC). In the 12 pancreatic cell lines analyzed, except for Capan-1 and Miapaca-2 where the level of αA-crystallin was about 80% and 65% of that in the control cell line, HPNE, the remaining pancreatic cancer cells have much lower αA-crystallin levels. Overexpression of αA-crystallin in MiaPaca-1 cells lacking endogenous αA-crystallin significantly decreased its tumorigenicity ability as shown in the colony formation and wound healing assays. In contrast, knockdown of αA-crystallin in the Capan-1 cells significantly increased its tumorigenicity ability as demonstrated in the above assays. Together, our results further demonstrate that αA-crystallin negatively regulates pancreatic tumorigenesis and appears to be a prognosis biomarker for PDAC.