Deuterium MRI of serine metabolism in mouse models of glioblastoma.

PURPOSE: Serine is a major source of one-carbon units needed for the synthesis of nucleotides and the production of intramitochondrial nicotinamide adenine dinucleotide phosphate (NADPH), and it plays an important role in cancer cell proliferation. The aim of this study was to develop a deuterium (2H) MRS imaging method for imaging tumor serine metabolism. METHODS: Sequential (2H) spectra and spectroscopic images were used to monitor the metabolism of [2,3,3-2H3]serine in patient-derived glioblastoma cells in vitro and in tumors obtained by their orthotopic implantation in mouse brain. RESULTS: [14,14-2H2] 5,10-methylene-tetrahydrofolate, [2H]glycine, [2H]formate, and labeled water were detected in cell suspensions and water labeling in spectroscopic images of tumors. Studies in cells and tumors with variable mitochondrial content and inhibitor studies in cells demonstrated that most of the labeled serine was metabolized in the mitochondria. Water labeling in the cell suspensions was correlated with formate labeling; therefore, water labeling observed in tumors could be used to provide a surrogate measure of flux in the pathway of one-carbon metabolism in vivo. CONCLUSION: The method has the potential to be used clinically to select patients for treatment with inhibitors of one-carbon metabolism and subsequently to detect their early responses to such treatment.

circBRAF promotes the progression of triple-negative breast cancer through modulating methylation by recruiting KDM4B to histone H3K9me3 and IGF2BP3 to mRNA.

Understanding the molecular characteristics of triple-negative breast cancer (TNBC) and developing more tailored treatment approaches is crucial. Circular RNAs (circRNAs), as potential therapeutic targets, remain largely unexplored in TNBC. This study utilized circRNA microarray analysis to determine the expression of circRNAs in TNBC, analyzing nine patient specimens. The characteristics of circBRAF were examined using divergent PCR primers, Sanger sequencing, fluorescence in situ hybridization (FISH) analysis, and the application of RNase and actinomycin D. The biological function of circBRAF in TNBC was further investigated through colony formation, tube formation, and transwell assays. Crucially, the mechanisms underlying the effects of circBRAF on TNBC progression were explored via RNA immunoprecipitation sequencing (RIP-seq) data, MS2 pulldown, RNA sequencing (RNA-seq) analysis, circBRAF knockdown, histone H3K9me3 modification, and Chromatin Isolation by RNA Purification (ChIRP) tests followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We focused particularly on hsa_circ_0007178, produced from exons 4-13 of the oncogene BRAF. Functional experiments revealed that circBRAF is crucial for the development of TNBC, with its knockdown preventing angiogenesis, metastasis, and cell division in vitro. Mechanistically, circBRAF interacts with KDM4B and IGF2BP3, promoting TNBC growth. Interaction of circBRAF with IGF2BP3 increased the expression of VCAN, FN1, CDCA3, or B4GALT3 by controlling mRNA stability through RNA N6-methyladenosine (m6A) modification. Furthermore, circBRAF upregulated the expression of ADAMTS14 and MMP9 through recruitment of KDM4B to enhance respective H3K9me3 modification. Furthermore, overexpression of circBRAF was able to overcome the inhibitory effects of siKDM4B and siIGF2BP3 on cell migration and invasion. Our findings suggest that circBRAF may act as an oncogene in TNBC through its specific interactions with KDM4B and IGF2BP3, implying that circBRAF could serve as a potentially effective novel therapeutic target for TNBC.

Deuterium Metabolic Imaging Differentiates Glioblastoma Metabolic Subtypes and Detects Early Response to Chemoradiotherapy.

Metabolic subtypes of glioblastoma (GBM) have different prognoses and responses to treatment. Deuterium metabolic imaging with 2H-labeled substrates is a potential approach to stratify patients into metabolic subtypes for targeted treatment. In this study, we used 2H magnetic resonance spectroscopy and magnetic resonance spectroscopic imaging (MRSI) measurements of [6,6'-2H2]glucose metabolism to identify metabolic subtypes and their responses to chemoradiotherapy in patient-derived GBM xenografts in vivo. The metabolism of patient-derived cells was first characterized in vitro by measuring the oxygen consumption rate, a marker of mitochondrial tricarboxylic acid cycle activity, as well as the extracellular acidification rate and 2H-labeled lactate production from [6,6'-2H2]glucose, which are markers of glycolytic activity. Two cell lines representative of a glycolytic subtype and two representative of a mitochondrial subtype were identified. 2H magnetic resonance spectroscopy and MRSI measurements showed similar concentrations of 2H-labeled glucose from [6,6'-2H2]glucose in all four tumor models when implanted orthotopically in mice. The glycolytic subtypes showed higher concentrations of 2H-labeled lactate than the mitochondrial subtypes and normal-appearing brain tissue, whereas the mitochondrial subtypes showed more glutamate/glutamine labeling, a surrogate for tricarboxylic acid cycle activity, than the glycolytic subtypes and normal-appearing brain tissue. The response of the tumors to chemoradiation could be detected within 24 hours of treatment completion, with the mitochondrial subtypes showing a decrease in both 2H-labeled glutamate/glutamine and lactate concentrations and glycolytic tumors showing a decrease in 2H-labeled lactate concentration. This technique has the potential to be used clinically for treatment selection and early detection of treatment response. SIGNIFICANCE: Deuterium magnetic resonance spectroscopic imaging of glucose metabolism has the potential to differentiate between glycolytic and mitochondrial metabolic subtypes in glioblastoma and to evaluate early treatment responses, which could guide patient treatment.

Mast cell degranulation-triggered by SARS-CoV-2 induces tracheal-bronchial epithelial inflammation and injury.

SARS-CoV-2 infection-induced hyper-inflammation is a key pathogenic factor of COVID-19. Our research, along with others', has demonstrated that mast cells (MCs) play a vital role in the initiation of hyper-inflammation caused by SARS-CoV-2. In previous study, we observed that SARS-CoV-2 infection induced the accumulation of MCs in the peri-bronchus and bronchioalveolar-duct junction in humanized mice. Additionally, we found that MC degranulation triggered by the spike protein resulted in inflammation in alveolar epithelial cells and capillary endothelial cells, leading to subsequent lung injury. The trachea and bronchus are the routes for SARS-CoV-2 transmission after virus inhalation, and inflammation in these regions could promote viral spread. MCs are widely distributed throughout the respiratory tract. Thus, in this study, we investigated the role of MCs and their degranulation in the development of inflammation in tracheal-bronchial epithelium. Histological analyses showed the accumulation and degranulation of MCs in the peri-trachea of humanized mice infected with SARS-CoV-2. MC degranulation caused lesions in trachea, and the formation of papillary hyperplasia was observed. Through transcriptome analysis in bronchial epithelial cells, we found that MC degranulation significantly altered multiple cellular signaling, particularly, leading to upregulated immune responses and inflammation. The administration of ebastine or loratadine effectively suppressed the induction of inflammatory factors in bronchial epithelial cells and alleviated tracheal injury in mice. Taken together, our findings confirm the essential role of MC degranulation in SARS-CoV-2-induced hyper-inflammation and the subsequent tissue lesions. Furthermore, our results support the use of ebastine or loratadine to inhibit SARS-CoV-2-triggered degranulation, thereby preventing tissue damage caused by hyper-inflammation.

PRMT5-mediated homologous recombination repair is essential to maintain genomic integrity of neural progenitor cells.

Maintaining genomic stability is a prerequisite for proliferating NPCs to ensure genetic fidelity. Though histone arginine methylation has been shown to play important roles in safeguarding genomic stability, the underlying mechanism during brain development is not fully understood. Protein arginine N-methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase that plays a role in transcriptional regulation. Here, we identify PRMT5 as a key regulator of DNA repair in response to double-strand breaks (DSBs) during NPC proliferation. Prmt5F/F; Emx1-Cre (cKO-Emx1) mice show a distinctive microcephaly phenotype, with partial loss of the dorsal medial cerebral cortex and complete loss of the corpus callosum and hippocampus. This phenotype is resulted from DSBs accumulation in the medial dorsal cortex followed by cell apoptosis. Both RNA sequencing and in vitro DNA repair analyses reveal that PRMT5 is required for DNA homologous recombination (HR) repair. PRMT5 specifically catalyzes H3R2me2s in proliferating NPCs in the developing mouse brain to enhance HR-related gene expression during DNA repair. Finally, overexpression of BRCA1 significantly rescues DSBs accumulation and cell apoptosis in PRMT5-deficient NSCs. Taken together, our results show that PRMT5 maintains genomic stability by regulating histone arginine methylation in proliferating NPCs.

Impact of Arterial Input Function and Pharmacokinetic Models on DCE-MRI Biomarkers for Detection of Vascular Effect Induced by Stroma-Directed Drug in an Orthotopic Mouse Model of Pancreatic Cancer.

PURPOSE: We demonstrated earlier in mouse models of pancreatic ductal adenocarcinoma (PDA) that Ktrans derived from dynamic contrast-enhanced (DCE) MRI detected microvascular effect induced by PEGPH20, a hyaluronidase which removes stromal hyaluronan, leading to reduced interstitial fluid pressure in the tumor (Clinical Cancer Res (2019) 25: 2314-2322). How the choice of pharmacokinetic (PK) model and arterial input function (AIF) may impact DCE-derived markers for detecting such an effect is not known. PROCEDURES: Retrospective analyses of the DCE-MRI of the orthotopic PDA model are performed to examine the impact of individual versus group AIF combined with Tofts model (TM), extended-Tofts model (ETM), or shutter-speed model (SSM) on the ability to detect the microvascular changes induced by PEGPH20 treatment. RESULTS: Individual AIF exhibit a marked difference in peak gadolinium concentration. However, across all three PK models, kep values show a significant correlation between individual versus group-AIF (p < 0.01). Regardless individual or group AIF, when kep is obtained from fitting the DCE-MRI data using the SSM, kep shows a significant increase after PEGPH20 treatment (p < 0.05 compared to the baseline); %change of kep from baseline to post-treatment is also significantly different between PEGPH20 versus vehicle group (p < 0.05). In comparison, when kep is derived from the TM, only the use of individual AIF leads to a significant increase of kep after PEGPH20 treatment, whereas the %change of kep is not different between PEGPH20 versus vehicle group. Group AIF but not individual AIF allows detection of a significant increase of Vp (derived from the ETM) in PEGPH20 versus vehicle group (p < 0.05). Increase of Vp is consistent with a large increase of mean capillary lumen area estimated from immunostaining. CONCLUSION: Our results suggest that kep derived from SSM and Vp from ETM, both using group AIF, are optimal for the detection of microvascular changes induced by stroma-directed drug PEGPH20. These analyses provide insights in the choice of PK model and AIF for optimal DCE protocol design in mouse pancreatic cancer models.

Role of ADAM33 short isoform as a tumor suppressor in the pathogenesis of thyroid cancer via oncogenic function disruption of full-length ADAM33.

Thyroid cancer is the most prevalent endocrine malignancy globally; however, its underlying pathogenesis remains unclarified. Reportedly, alternative splicing is involved in processes such as embryonic stem and precursor cell differentiation, cell lineage reprogramming, and epithelial-mesenchymal transitions. ADAM33-n, an alternative splicing isoform of ADAM33, encodes a small protein containing 138 amino acids of the N-terminal of full-length ADAM33, which constructs a chaperone-like domain that was previously reported to bind and block the proteolysis activity of ADAM33. In this study, we reported for the first time that ADAM33-n was downregulated in thyroid cancer. The results of cell counting kit-8 and colony formation assays showed that ectopic ADAM33-n in papillary thyroid cancer cell lines restricted cell proliferation and colony formation. Moreover, we demonstrated that ectopic ADAM33-n reversed the oncogenic function of full-length ADAM33 in cell growth and colony formation in the MDA-T32 and BCPAP cells. These findings indicate the tumor suppressor ability of ADAM33-n. Altogether, our study findings present a potential explanatory model of how the downregulation of the oncogenic gene ADAM33 promotes the pathogenesis of thyroid cancer.

The cytoplasmic synthesis and coupled membrane translocation of eukaryotic polyphosphate by signal-activated VTC complex.

Inorganic polyphosphate (polyP) is an ancient energy metabolite and phosphate store that occurs ubiquitously in all organisms. The vacuolar transporter chaperone (VTC) complex integrates cytosolic polyP synthesis from ATP and polyP membrane translocation into the vacuolar lumen. In yeast and in other eukaryotes, polyP synthesis is regulated by inositol pyrophosphate (PP-InsP) nutrient messengers, directly sensed by the VTC complex. Here, we report the cryo-electron microscopy structure of signal-activated VTC complex at 3.0 Å resolution. Baker's yeast VTC subunits Vtc1, Vtc3, and Vtc4 assemble into a 3:1:1 complex. Fifteen trans-membrane helices form a novel membrane channel enabling the transport of newly synthesized polyP into the vacuolar lumen. PP-InsP binding orients the catalytic polymerase domain at the entrance of the trans-membrane channel, both activating the enzyme and coupling polyP synthesis and membrane translocation. Together with biochemical and cellular studies, our work provides mechanistic insights into the biogenesis of an ancient energy metabolite.

18F-Fluciclovine PET Imaging of Glutaminase Inhibition in Breast Cancer Models.

Aggressive cancers such as triple-negative breast cancer (TNBC) avidly metabolize glutamine as a feature of their malignant phenotype. The conversion of glutamine to glutamate by the glutaminase enzyme represents the first and rate-limiting step of this pathway and a target for drug development. Indeed, a novel glutaminase inhibitor (GLSi) has been developed and tested in clinical trials but with limited success, suggesting the potential for a biomarker to select patients who could benefit from this novel therapy. Here, we studied a nonmetabolized amino acid analog, 18F-fluciclovine, as a PET imaging biomarker for detecting the pharmacodynamic response to GLSi. Methods: Uptake of 18F-fluciclovine into human breast cancer cells was studied in the presence and absence of inhibitors of glutamine transporters and GLSi. To allow 18F-fluciclovine PET to be performed on mice, citrate in the tracer formulation is replaced by phosphate-buffered saline. Mice bearing triple-negative breast cancer (TNBC) xenografts (HCC38, HCC1806, and MBA-MD-231) and estrogen receptor-positive breast cancer xenografts (MCF-7) were imaged with dynamic PET at baseline and after a 2-d treatment of GLSi (CB839) or vehicle. Kinetic analysis suggested reversible uptake of the tracer, and the distribution volume (VD) of 18F-fluciclovine was estimated by Logan plot analysis. Results: Our data showed that cellular uptake of 18F-fluciclovine is mediated by glutamine transporters. A significant increase in VD was observed after CB839 treatment in TNBC models exhibiting high glutaminase activity (HCC38 and HCC1806) but not in TNBC or MCF-7 exhibiting low glutaminase. Changes in VD were corroborated with changes in GLS activity measured in tumors treated with CB839 versus vehicle, as well as with changes in VD of 18F-(2S,R4)-fluoroglutamine, which we previously validated as a measure of cellular glutamine pool size. A moderate, albeit significant, decrease in 18F-FDG PET signal was observed in HCC1806 tumors after CB839 treatment. Conclusion: 18F-fluciclovine PET has potential to serve as a clinically translatable pharmacodynamic biomarker of GLSi.

DWI Metrics Differentiating Benign Intraductal Papillary Mucinous Neoplasms from Invasive Pancreatic Cancer: A Study in GEM Models.

KPC (KrasG12D:Trp53R172H:Pdx1-Cre) and CKS (KrasG12D:Smad4L/L:Ptf1a-Cre) mice are genetically engineered mouse (GEM) models that capture features of human pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasms (IPMN), respectively. We compared these autochthonous tumors using quantitative imaging metrics from diffusion-weighted MRI (DW-MRI) and dynamic contrast enhanced (DCE)-MRI in reference to quantitative histological metrics including cell density, fibrosis, and microvasculature density. Our results revealed distinct DW-MRI metrics between the KPC vs. CKS model (mimicking human PDAC vs. IPMN lesion): the apparent diffusion coefficient (ADC) of CKS tumors is significantly higher than that of KPC, with little overlap (mean ± SD 2.24±0.2 vs. 1.66±0.2, p<10−10) despite intratumor and intertumor variability. Kurtosis index (KI) is also distinctively separated in the two models. DW imaging metrics are consistent with growth pattern, cell density, and the cystic nature of the CKS tumors. Coregistration of ex vivo ADC maps with H&E-stained sections allowed for regional comparison and showed a correlation between local cell density and ADC value. In conclusion, studies in GEM models demonstrate the potential utility of diffusion-weighted MRI metrics for distinguishing pancreatic cancer from benign pancreatic cysts such as IPMN.

Effect of berbamine on invasion and metastasis of human liver cancer SMMC-7721 cells and its possible mechanism.

Berbamine is a bisbenzylisoquinoline alkaloid extracted from Berberis poiretii of Berberis of Berberidaceae. It has been reported that it can significantly inhibit the proliferation of a variety of malignant tumor cells, including liver cancer. However, the effect of berbamine on the invasion and metastasis of liver cancer has not been reported. The present study demonstrated that berbamine inhibited the migration and invasion of SMMC-7721 cells in a concentration-dependent manner and obviously increased the gap junction function and the expression of Cx32 in SMMC-7721 cells compared with control group. However, after silencing Cx32, berbamine had no significant effect on cell invasion and metastasis. Before silencing Cx32, the expression of PI3K and P-AKT were decreased after berbamine treated on SMMC-7721 cells for 24 h. After silencing Cx32, the expression of PI3K and P-AKT were increased in SMMC-7721 cells. The expression of PI3K and P-AKT had no significant effect after berbamine treated on SMMC-7721 cells for 24 h with silencing Cx32. In conclusion, the results of the present study suggest that berbamine could inhibit the SMMC-7721 cell migration and invasion, and its mechanism may be related to the regulation of PI3K/AKT signaling pathway by enhancing the expression of Cx32.

The ctDNA-based postoperative molecular residual disease status in different subtypes of early-stage breast cancer.

Background: Breast cancer is a highly heterogeneous disease. Early-stage, non-metastatic breast cancer is considered curable after definitive treatment. Early detection of tumor recurrence and metastasis through sensitive biomarkers is helpful for guiding clinical decision-making and early intervention in second-line treatment, which could improve patient prognosis and survival. Methods: In this real-world study, we retrospectively analyzed 82 patients with stages I to III breast cancer who had been analyzed by molecular residual disease (MRD) assay. A total of 82 tumor tissues and 224 peripheral blood samples were collected and detected by next-generation sequencing (NGS) based on a 1,021-gene panel in this study. Results: MRD positivity was detected in 18 of 82 patients (22.0%). The hormone receptor-/human epidermal growth factor receptor 2+ (HR-/HER2+) subgroup had the highest postoperative MRD detection rate at 30.8% (4/13). The BRCA2 and SLX4 genes were significantly enriched in all patients in the MRD positive group and FGFR1 amplification was significantly enriched in the MRD negative group with HR+/HER2-. The number of single nucleotide variants (SNVs) in tissue samples of MRD-positive patients was higher than that of MRD-negative patients (11.94 vs. 8.50 SNVs/sample). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that there was a similar biological function of the tumor-mutated genes in the 2 MRD status groups. Conclusions: This real-world study confirmed that patient samples of primary tumor tissue with different MRD status and molecular subtypes had differential genetic features, which may be used to predict patients at high risk for recurrence.

A Novel Netrin-1-Derived Peptide Enhances Protection against Neuronal Death and Mitigates of Intracerebral Hemorrhage in Mice.

It has been reported that Netrin-1 is involved in neuroprotection following injury to the central nervous system. However, the minimal functional domain of Netrin-1 which can preserve the neuroprotection but avoid the major side effects of Netrin remains elusive. Here, we investigated the neuroprotective effect of a peptide E1 derived from Netrin-1's EGF3 domain (residues 407-422). We found that it interacts with deleted colorectal carcinoma (DCC) to activate focal adhesion kinase phosphorylation exhibiting neuroprotection. The administration of the peptide E1 was able to improve functional recovery through reduced apoptosis in an experimental murine model of intracerebral hemorrhage (ICH). In summary, we reveal a functional sequence of Netrin-1 that is involved in the recovery process after ICH and identify a candidate peptide for the treatment of ICH.

Respiratory Motion Mitigation and Repeatability of Two Diffusion-Weighted MRI Methods Applied to a Murine Model of Spontaneous Pancreatic Cancer.

Respiratory motion and increased susceptibility effects at high magnetic fields pose challenges for quantitative diffusion-weighted MRI (DWI) of a mouse abdomen on preclinical MRI systems. We demonstrate the first application of radial k-space-sampled (RAD) DWI of a mouse abdomen using a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) on a 4.7 T preclinical scanner equipped with moderate gradient capability. RAD DWI was compared with the echo-planar imaging (EPI)-based DWI method with similar voxel volumes and acquisition times over a wide range of b-values (0.64, 535, 1071, 1478, and 2141 mm2/s). The repeatability metrics are assessed in a rigorous test-retest study (n = 10 for each DWI protocol). The four-shot EPI DWI protocol leads to higher signal-to-noise ratio (SNR) in diffusion-weighted images with persisting ghosting artifacts, whereas the RAD DWI protocol produces relatively artifact-free images over all b-values examined. Despite different degrees of motion mitigation, both RAD DWI and EPI DWI allow parametric maps of apparent diffusion coefficients (ADC) to be produced, and the ADC of the PDAC tumor estimated by the two methods are 1.3 ± 0.24 and 1.5 ± 0.28 × 10-3 mm2/s, respectively (p = 0.075, n = 10), and those of a water phantom are 3.2 ± 0.29 and 2.8 ± 0.15 × 10-3 mm2/s, respectively (p = 0.001, n = 10). Bland-Altman plots and probability density function reveal good repeatability for both protocols, whose repeatability metrics do not differ significantly. In conclusion, RAD DWI enables a more effective respiratory motion mitigation but lower SNR, while the performance of EPI DWI is expected to improve with more advanced gradient hardware.

Dynamic Contrast-enhanced MRI Detects Responses to Stroma-directed Therapy in Mouse Models of Pancreatic Ductal Adenocarcinoma.

PURPOSE: The dense stroma underlies the drug resistance of pancreatic ductal adenocarcinoma (PDA) and has motivated the development of stroma-directed drugs. Our objective is to test the concept that dynamic contrast-enhanced (DCE) MRI using FDA-approved contrast media, an imaging method sensitive to the tumor microenvironment, can detect early responses to stroma-directed drug. EXPERIMENTAL DESIGN: Imaging studies were performed in three mouse models exhibiting high desmoplastic reactions: the autochthonous PDA in genetically engineered mice (KPC), an orthotopic model in syngeneic mice, and a xenograft model of human PDA in athymic mice. An investigational drug, PEGPH20 (pegvorhyaluronidase alfa), which degrades hyaluronan (HA) in the stroma of PDA, was injected alone or in combination with gemcitabine. RESULTS: At 24 hours after a single injection of PEGPH20, Ktrans , a DCE-MRI-derived marker that measures how fast a unit volume of contrast media is transferred from capillaries to interstitial space, increased 56% and 50% from baseline in the orthotopic and xenograft tumors, respectively, compared with a 4% and 6% decrease in vehicle groups (both P < 0.05). Similarly, after three combined treatments, Ktrans in KPC mice increased 54%, whereas it decreased 4% in controls treated with gemcitabine alone (P < 0.05). Consistently, after a single injection of PEGPH20, tumor HA content assessed by IHC was reduced substantially in all three models while drug delivery (measured by paclitaxel accumulation in tumor) was increased by 2.6-fold. CONCLUSIONS: These data demonstrated a DCE-MRI marker, Ktrans , can detect early responses to stroma-directed drug and reveal the sustained effect of combination treatment (PEGPH20+ gemcitabine).

Berbamine induces SMMC-7721 cell apoptosis via upregulating p53, downregulating survivin expression and activating mitochondria signaling pathway.

Hepatocellular carcinoma (HCC) is a primary malignancy in the liver, which is a global health problem. The present study aimed to observe the apoptotic effects of berbamine on SMMC-7721 cell lines and to investigate the effects of berbamine on induction of the intrinsic apoptotic pathway. The human HCC SMMC-7721 cells were cultured and cell morphology observed using a phase contrast microscope. SMMC-7721 cell apoptosis was examined by employing a flow cytometry assay. The nuclei of SMMC-7721 cells were stained with DAPI and observed by utilizing a laser fluorescence microscope. Cytochrome c (Cyto c) levels were evaluated by using immunofluorescence staining. The reverse transcription-semi-quantitative polymerase chain reaction (RT-sqPCR) and western blot analysis were used to examine the mRNA and protein levels of B-cell lymphoma 2, (Bcl-2), Bax, Bcl-2-associated X, apoptosis regulator, p53 and survivin, respectively. Berbamine inhibited SMMC-7721 cell growth at 20 and 0 µmol/l, compared with control group (0 µmol/l berbamine). DAPI results demonstrated that berbamine affected the nucleus morphology of SMMC-7721 cells. Berbamine at a concentration of 20 µmol/l (P<0.05) and 40 µmol/l (P<0.01) significantly enhanced apoptosis rate compared with control group. Berbamine triggered Cyto c release from SMMC-7721 cell nuclei to the cytoplasm. Berbamine (10, 20, 40 µmol/l) significantly enhanced Bax and p53 levels and decreased Bcl-2 and survivin levels compared with control group, according to RT-sqPCR and western blot assay findings. In conclusion, berbamine induced SMMC-7721 cell apoptosis, through upregulating p53 expression and downregulating survivin expression, which further triggered mitochondria signaling pathway-mediated apoptosis.

In Vivo Magnetic Resonance and Fluorescence Dual-Modality Imaging of Tumor Angiogenesis in Rats Using GEBP11 Peptide Targeted Magnetic Nanoparticles.

Angiogenesis is an essential process for tumor progression. Tumor vasculature-targeting peptides have shown great potential for use in cancer imaging and therapy. Our previous studies have shown that GEBP11, a novel vasculature-specific binding peptide that exhibits high affinity and specificity to tumor angiogenesis, is a promising candidate for the diagnosis and targeted radiotherapy of gastric cancer. In the present study, we developed a novel magnetic resonance and fluorescence (MR/Fluo) dual-modality imaging probe by covalently coupling 2,3-dimercaptosuccinnic acid-coated paramagnetic nanoparticles (DMSA-MNPs) and Cy5.5 to the GEBP11 peptide. The probe Cy5.5-GEBP11-DMSA-MNPs (CGD-MNPs), with a hydrodynamic diameter of 82.8 ± 6.5 nm, exhibited good imaging properties, high stability and little cytotoxicity. In vivo MR/Fluo imaging revealed that CGD-MNPs were successfully applied to visualize tumor angiogenesis in SGC-7901 xenograft mouse models. Prussian blue and CD31 immunohistochemical staining confirmed that CGD-MNPs co-localized with tumor blood vessels. In conclusion, CGD-MNPs are promising candidates for use as MR and fluorescence imaging probes for visualizing gastric cancer angiogenesis in vivo.

Monitoring of the tumor response to nano-graphene oxide-mediated photothermal/photodynamic therapy by diffusion-weighted and BOLD MRI.

Photothermal therapy (PTT) and photodynamic therapy (PDT) are promising cancer treatment modalities. Because each modality has its own set of advantages and limitations, there has been interest in developing methods that can co-deliver the two regimens for enhanced tumor treatment. Among the efforts, nano-graphene oxide-mediated phototherapies have recently attracted much attention. Nano-graphene oxide has a broad absorbance spectrum and can be loaded with photosensitizers, such as chlorin e6, with high efficiency. Chlorin e6-loaded and PEGylated nano-graphene (GO-PEG-Ce6) can be excited at 660 nm, 808 nm, or both, to induce PDT, PTT, or PDT/PTT combination. Despite the potential of the treatments, there is a lack of a diagnostic tool which can monitor their therapeutic response in a non-invasive and prognostic manner; such an ability is urgently needed for the transformation and translation of the technologies. In this study, we performed diffusion-weighted and blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) after GO-PEG-Ce6-mediated PTT, PDT, or PTT/PDT. We found that after efficient PTT, there is a significant increase of the tumor apparent diffusion coefficient (ADC) value in diffusion-weighted imaging (DWI) maps; meanwhile, an efficient PDT led to an increase of in BOLD images. In both the cases, the amplitude of the increase was correlated with the treatment outcomes. More interestingly, a synergistic treatment efficacy was observed when the PTT/PDT combination was applied, and the combination was associated with a greater ADC and increase than when either modality was used alone. In particular, the PTT/PDT condition that induced the most dramatic short-term increase of the ADC value (>70%) caused the most effective tumor control in the long-run, with 60% of the treated animals being tumor-free after 60 days. These results suggest the great promise of the combination of DWI and BOLD MRI as a tool for accurate monitoring and prognosis of phototherapies, which is of great value to the future developments of the methodologies.

Noninvasive Dynamic Imaging of Tumor Early Response to Nanoparticle-mediated Photothermal Therapy.

In spite of rapidly increasing interest in the use of nanoparticle-mediated photothermal therapy (PTT) for treatment of different types of tumors, very little is known on early treatment-related changes in tumor response. Using graphene oxide (GO) as a model nanoparticle (NP), in this study, we tracked the changes in tumors after GO NP-mediated PTT by magnetic resonance imaging (MRI) and quantitatively identified MRI multiple parameters to assess the dynamic changes of MRI signal in tumor at different heating levels and duration. We found a time- and temperature-dependent dynamic change of the MRI signal intensity in intratumor microenvironment prior to any morphological change of tumor, mainly due to quick and effective eradication of tumor blood vessels. Based on the distribution of GO particles, we also demonstrated that NP-medited PTT caused heterogeneous thermal injury of tumor. Overall, these new findings provide not only a clinical-related method for non-invasive early tracking, identifying, and monitoring treatment response of NP-mediated PTT but also show a new vision for better understanding mechanisms of NP-mediated PTT.

Co9 Se8 nanoplates as a new theranostic platform for photoacoustic/magnetic resonance dual-modal-imaging-guided chemo-photothermal combination therapy.

A new theranostic platform is developed based on biocompatible poly(acrylic acid) (PAA)-Co9 Se8 nanoplates. These PAA-Co9 Se8 nanoplates are successfully utilized for photoacoustic imaging (PAI)/magnetic resonance imaging (MRI) dual-modal imaging. Moreover, the PAA-Co9 Se8 -DOX shows pH-responsive chemotherapy and enables the combination of photothermal therapy and chemotherapy to receive superior antitumor efficacy. This work promises further exploration of 2D nanoplatforms for theranostic applications.