A PARP1 PROTAC as a novel strategy against PARP inhibitor resistance via promotion of ferroptosis in p53-positive breast cancer.

Therapeutic targeting of the nuclear enzyme poly (ADP-ribose) polymerase 1 (PARP1) with PARP inhibitors (PARPis) in patients with a homologous recombination (HR)- deficient phenotype based on the mechanism of synthetic lethality has been shown tremendous success in cancer therapy. With the clinical use of various PARPis, emerging evidence has shown that some PARPis offer hope for breakthroughs in triple-negative breast cancer (TNBC) therapy, regardless of HR status. However, similar to other conventional cytotoxic drugs, PARPis are also subject to the intractable problem of drug resistance. Notably, acquired resistance to PARPis caused by point mutations in the PARP1 protein is hard to overcome with current strategies. To explore modalities to overcome resistance and identify patients who are most likely to benefit from PARP1-targeted therapy, we developed a proteolysis-targeted chimaera (PROTAC) to degrade mutant PARP1 in TNBC. Here, we investigated a PARP1 PROTAC termed "NN3″, which triggered ubiquitination and proteasome-mediated degradation of PARP1. Moreover, NN3 degraded PARP1 with resistance-related mutations. Interestingly, compared with other reported PARP1 degraders, NN3 exhibited a unique antitumor mechanism in p53-positive breast cancer cells that effectively promoted ferroptosis by downregulating the SLC7A11 pathway. Furthermore, NN3 showed potent activity and low toxicity in vivo. In conclusion, we propose PROTAC-mediated degradation of PARP1 as a novel strategy against mutation-related PARPi resistance and a paradigm for targeting breast cancer with functional p53 via ferroptosis induction.

The Radiological Imaging Features of Easily Misdiagnosed Epithelioid Glioblastoma in Seven Patients.

OBJECTIVE: We review the radiological imaging features and report histopathological findings of 7 adult patients with epithelioid glioblastoma (eGBM), which was a newly revised subtype of glioblastoma. METHODS: Seven adult patients with a diagnosis of eGBM on a brain tissue specimen were retrospectively confirmed by pathology. The tumor magnetic resonance imaging characteristics such as location, number, edema, necrosis, hemorrhage, enhancement, diffusion-weighted image, apparent diffusion coefficient, magnetic resonance spectroscopy, dynamic susceptibility contrast-perfusion-weighted imaging, and histopathological findings were documented. RESULTS: The tumors of these patients exhibited iso-hyperintensive signal on the T2-weighted image and iso-hypointensive signal on the T1-weighted image. All the lesions manifested iso-hypointensive signal on the diffusion-weighted image, and 2 cases showed significantly restricted hyperintensive signal (2/7). Peritumoral edema in all cases was mild. Five cases were located in the cortical lobe (5/7) and the other 2 were multifocal (2/7). Four cases showed white matter collapse sign (4/7). These tumors revealed apparent enhancement after contrast injection. In particular, 4 cases displayed capsuled sign (4/7), 2 cases showed dura mater tail sign (2/7), and 1 case showed hemorrhage (1/7). The mean value of apparent diffusion coefficient in 6 cases was 1.09 × 10-3 mm2/s. The mean value of relative cerebral blood volume in 3 cases was 2.84 × 10-3 mm2/s on dynamic susceptibility contrast-perfusion-weighted imaging. The average value of the choline/N-acetyl aspartate ratio in 4 cases was 6.47 × 10-3 mm2/s on magnetic resonance spectroscopy. All cases expressed a BRAF V600E mutation according to molecular characteristics. CONCLUSIONS: eGBMs that were predominantly located in cortex with mild peritumoral edema, white matter collapse, encapsular sign, and dura mater tail sign could be easily misdiagnosed as cortex-involved intracranial brain tumor such as meningioma, whereas multifocal tumors could be easily misdiagnosed as metastatic tumor and lymphoma. Multimodal images were helpful for the differential diagnosis.

PA28ß regulates cell invasion of gastric cancer via modulating the expression of chloride intracellular channel 1.

PA28ß is a subunit of proteasome activator PA28. Previous study suggests that PA28ß is involved in the invasiveness and metastasis of gastric adenocarcinoma (GA), however, the mechanism is not fully understood. In the present study, we showed that invasive abilities of gastric cancer cells were enhanced when PA28ß being down-regulated, and were inhibited when PA28ß being overexpressed. To explore the possible mechanism of PA28ß associated elevated invasiveness, the protein profiles of PA28ß knock down and parental negative control gastric cancer cells were compared using proteomics approach. The results revealed that there were 43 proteins were differentially expressed, among them, chloride intracellular channel 1 (CLIC1) was significantly up-regulated and selected for further functional study. Down-regulation of CLIC1 by RNA interference was able to markedly inhibit cell invasion of PA28ß knock down gastric carcinoma cells. In addition, an inverse correlation between PA28ß and CLIC1 expressions was also verified in GA tissue samples, suggesting that knockdown of PA28ß could enhance tumor invasion and metastasis, at least in part, through up-regulation of CLIC1. Our results provide novel insight into the mechanisms of PA28ß related invasiveness and metastasis of GA, and suggest new alternative approaches for GA treatment.

Notch-4 contributes to the metastasis of salivary adenoid cystic carcinoma.

The Notch signaling pathway is important for cell-cell communication; it is involved in gene regulation mechanisms that control multiple cell differentiation processes during embryonic and adult life. Notch is present in all metazoans, and vertebrates possess four different Notch receptors: Notch-1, Notch-2, Notch-3, and Notch-4. The aim of the present study was to identify the role of Notch protein in the metastasis of salivary adenoid cystic carcinoma (SACC). Real-time PCR results showed that Notch-1, Notch-2, and Notch-4 were upregulated in the highly metastatic SACC cell line ACC-M, compared to ACC-2, a SACC cell line with low metastatic ability. Knockdown of Notch-4 by small interfering RNA efficiently inhibited the invasion of ACC-M cells. Notch-4 expression was significantly higher in the clinical samples with metastasis and recurrence compared to that in control (p<0.05), shown by immunohistochemistry analysis. These results indicate that Notch-4 may play an important role in SACC metastasis.

PLC/CAMK IV-NF-kappaB involved in the receptor for advanced glycation end products mediated signaling pathway in human endothelial cells.

Advanced glycation end products (AGEs) and their interaction with the receptor for advanced glycation end products (RAGE) play an important role in diabetic vascular complications. The current study demonstrated that AGEs significantly increased RAGE expression and the release of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) in human umbilical vein endothelial cell-derived line ECV304 cells. RAGE antisense RNA partially inhibited the expression of TNF-alpha and IL-6 induced by AGEs. Oligonucleotide microarray was used to identify the genes that respond to RAGE activation. Phospholipase C beta 1 (PLC beta 1), phospholipase C beta 4 (PLC beta 4) and calcium/calmodulin-dependent protein kinase IV (CAMK IV) which associated with Ca(2+) signaling were upregulated. The rise of intracellular calcium and the NF-kappaB promoter activity induced by AGEs were suppressed by RAGE antisense RNA, PLC inhibitor U73122 and dominant negative CAMK IV, respectively. These findings suggest that PLC/CAMK IV-NF-kappaB is involved in RAGE mediated signaling pathway in human endothelial cells.

Biological impacts of "hot-spot" mutations of hepatitis B virus X proteins are genotype B and C differentiated.

AIM: To investigate the biological impacts of "hot-spot" mutations on genotype B and C HBV X proteins (HBx). METHODS: Five types of "hot-spot" mutations of genotype B or C HBV X genes, which sequentially lead to the amino acid substitutions of HBx as I127T, F132Y, K130M+V131I, I127T+K130M+V131I, or K130M+V131I+F132Y, respectively, were generated by means of site-directed mutagenesis. To evaluate the anti-proliferative effects, HBx or related mutants' expression vectors were transfected separately to the Chang cells by lipofectamine, and the cells were cultured in hygromycin selective medium for 14 d, drug-resistant colonies were fixed with cold methanol, stained with Giemsa dyes and scored (increase of the colonies indicated the reduction of the anti-proliferation activity, and vice versa). Different types of HBx expression vectors were co-transfected separately with the reporter plasmid pCMVbeta to Chang cells, which were lysed 48 h post-transfection and the intra-cellular beta-galactosidase activities were monitored (increase of the beta-galactosidase activities indicated the reduction of the transactivation activity, and vice versa). All data obtained were calculated by paired-samples t-test. RESULTS: As compared to standard genotype B HBx, mutants of I127T and I127T+K130M+V131I showed higher transactivation and anti-proliferative activities, while the mutants of F132Y, K130M+V131I, and K130M+V131I+F132Y showed lower activities. As compared to standard genotype C HBx, I127T mutant showed higher transactivation activity, while the other four types of mutants showed no differences. With regard to anti-proliferative activity, compared to standard genotype C HBx, F132Y and K130M+ V131I mutants showed lower activities, and K130M+V131I +F132Y mutant, on the other hand, showed higher activity, while the mutants of I127T and I127T+K130M+V131I showed no differences. CONCLUSION: "Hot-spot" mutations affect the anti-proliferation and transactivation activities of genotype B and/or C HBx, and the biological impacts of most "hot-spot" mutations on HBx are genotype B and C differentiated.

[X protein variations of genotype B and C hepatitis B virus isolated from the patients with hepatocellular carcinoma].

BACKGROUND & OBJECTIVE: X protein was one of the most important pathogens of hepatitis B virus (HBV), and was crucial in the carcinogenesis of HBV related hepatocellular carcinoma (HCC). It was demonstrated that the infection of genotype B or C HBV would result in different clinical manifestations and pathological characteristics in HCC patients, however, it was under elucidation whether these differences related to different genotypes of HBV X protein. This study was designed to investigate the amino acid differentiations of X proteins in standard genotype B or C HBV strains and the variations in HBV isolated from the HCC patients, and elucidate preliminarily the relationship between the genotype of HBV and carcinogenesis of HCC. METHODS: HBV X genes from the serum of twenty-two HBsAg positive HCC patients were amplified, cloned and sequenced. Genotyping of the X gene was carried out by Vector NTI6.0 software and the amino acid alignment among the standard and HCC originated X protein were done by DNAMAN software. RESULTS: Twenty-two HBV X genes were obtained and all of them could be categorized into genotype B or C. Besides of 14 amino acid differentiations within X protein between standard B and C HBV strains, HCC originated X protein of B and C genotype showed 4 consensus amino acid variations, and genotype C X protein showed additional 6 genotype-specific variations. All these differentiated and varied amino acids were located in the B, T cell epitopes and transactive or related regulatory regions. CONCLUSIONS: In addition to amino acid differentiations, X protein of genotype B or C HBV also showed genotype-specific variations in HCC patients. Amino acid differentiations and variations may result in the different immunocompetence and transactivation capacity between X proteins of genotype B and C.

Reflectins: the unusual proteins of squid reflective tissues.

A family of unusual proteins is deposited in flat, structural platelets in reflective tissues of the squid Euprymna scolopes. These proteins, which we have named reflectins, are encoded by at least six genes in three subfamilies and have no reported homologs outside of squids. Reflectins possess five repeating domains, which are highly conserved among members of the family. The proteins have a very unusual composition, with four relatively rare residues (tyrosine, methionine, arginine, and tryptophan) comprising approximately 57% of a reflectin, and several common residues (alanine, isoleucine, leucine, and lysine) occurring in none of the family members. These protein-based reflectors in squids provide a marked example of nanofabrication in animal systems.

[Cloning, expression and purification of the major surface antigen P30 of Toxoplasma gondii].

OBJECTIVE: To obtain protein of the major surface antigen P30 of Toxoplasma gondii by molecular cloning. METHODS: The gene of P30 (containing the whole P30 gene sequence, without the gene encoding signal peptide) was obtained by polymerase chain reaction (PCR) using the primer designed according to the DNA sequence of P30. The recombinant plasmid was constructed using EcoR I, Xho I and was then transformed into E. coli Top10. The positive clones were identified by restriction enzymes and DNA sequence analysis. The fusion protein was induced by IPTG and purified by affinity chromatography using ProBond Resin (a kind of nickel-charged sepharose resin) and was identified by SDS-PAGE and Western blotting. RESULTS: The products of PCR, cleavage and link reaction were same as expected and the sequence of inserted fragment in the recombinant plasmid was same as reported except one synonymy mutation. A 58 kDa fusion protein was induced by IPTG and was purified by chromatography. CONCLUSION: Fusion protein containing Toxoplasma gondii P30 was achieved and was provided as experiment material for further research.

[Expression and significance of nitric oxide synthase in human hepatocellular carcinoma].

OBJECTIVE: To investigate the expression and significance of nitric oxide synthase (cNOS) mRNA in primary hepatocellular carcinoma (HCC), cirrhotic liver and normal liver tissue. METHODS: cNOS mRNA expression in 80 HCC, 40 cirrhotic liver and 20 normal liver tissue were observed by in situ hybridization. CD34 immunostain was used to measure the microvascular density (MVD) and Ki-67 immunostain to proliferative index. RESULTS: Expression of cNOS mRNA was observed in the liver cancer cells, endothelial cells in the non-cancerous liver tissues and mononuclear and/or phagocytes. Expression of cNOS mRNA in tumor cells of HCC was higher than that in the liver cells of cirrhotic liver (P < 0.01) which was higher than the normal liver tissue. Expression in the endothelial cells was higher in HCC and cirrhotic liver than those in the normal liver tissue (P < 0.01). HCC with positive cNOS mRNA expression in the endothelial cells showed higher extent of neovascularization and degree of proliferative index. The more MVD, the higher proliferative index, which increased in metastatic tumors. CONCLUSION: cNOS mRNA expression was involved in oncogenesis, angiogenesis and progression of hepatocellular carcinoma.

[Expression of 67kD laminin receptor in human hepatocellular carcinoma cells].

BACKGROUND & OBJECTIVE: Previous study of our laboratory showed that 67kD laminin receptor (67LR) from SMMC-7721 hepatocellular carcinoma (HCC) cells had higher binding affinity with laminin than that from L-02 normal hepatic cells; however, the expression level of 67LR in HCC cells and normal hepatic cells was still unknown. The aim of this study was to investigate the relationship between laminin binding activity and the expression of mRNA and protein of 67kD laminin receptor in SMMC-7721,HepG2 HCC cells, and L-02 hepatic cells. METHODS: (1) The binding of laminin with three kinds of cells was quantified by (131)I-labeled laminin. (2)The expression of 67kD laminin receptor was determined by the reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. RESULTS: (1) Under the same condition, the amount of radiolabelled laminin bound to SMMC-7721, HepG2 HCC cells was 17.54+/-0.49 ng per 10(5) cells and 11.18+/-0.53 ng per 10(5) cells, respectively, which were significantly higher than that to L-02 hepatic cells (8.36+/-0.48 ng per 10(5) cells). The binding of laminin with HCC cells is specific and saturable, and the cells bound exogenous laminin with high affinity. (2) 67 LR showed notably increased expression on the surface of L-02 hepatic cells (55.3%), compared with SMMC-7721 HCC cells (34.7%) by flow cytometry. HepG2 did not express membrance 67 LR. (3) The expression of 67LR mRNA in SMMC-7721 and HepG2 HCC cells was higher than that in L-02 cells. CONCLUSION: The conflict of laminin binding activity and the expression of 67 LR in the three cell lines suggested SMMC-7721 HCC cells may express 67 LR with higher laminin affinity than L-02 hepatic cells, and other laminin receptors were involved in laminin binding.

[Signaling role of exogenous arachidonic acid in the invasion of macrophages by Toxoplasma gondii].

OBJECTIVE: To investigate the signaling role of arachidonic acid in the invasion of RAW264.7 macrophage by Toxoplasma gondii. METHODS: Rate of infection was calculated by both light microscope and flow cytometer. Fluorescent emission spectra were recorded using a microspectrofluometer for the concentration of cytoplasmic free calcium. RESULTS: Calcium concentration in macrophages and rate of infection increased with a higher concentration of exogenous arachidonic acid in a dose-dependent manner. The invasion was dependent on the mobilization of calcium from the extracellular medium and from intracellular stores and followed the influx of calcium into the parasitized cell. CONCLUSION: Arachidonic acid may enhance the rate of infection via calcium transduction pathway.