P2X7 Integrates PI3K/AKT and AMPK-PRAS40-mTOR Signaling Pathways to Mediate Tumor Cell Death

Background

Extracellular adenosine triphosphate (ATP) functions as a novel danger signal that boosts antitumor immunity and can also directly kill tumor cells. We have previously reported that chronic exposure of tumor cells to ATP provokes P2X7-mediated tumor cell death, by as yet incompletely defined molecular mechanisms.

Methodology/Principal Findings

Here, we show that acute exposure of tumor cells to ATP results in rapid cytotoxic effects impacting several aspects of cell growth/survival, leading to inhibition of tumor growth in vitro and in vivo. Using agonist and antagonist studies together with generation of P2X7 deficient tumor cell lines by lentiviral shRNA delivery system, we confirm P2X7 to be the central control node transmitting extracellular ATP signals. We identify that downstream intracellular signaling regulatory networks implicate two signaling pathways: the known P2X7-PI3K/AKT axis and remarkably a novel P2X7-AMPK-PRAS40-mTOR axis. When exposed to high levels of extracellular ATP, these two signaling axes perturb the balance between growth and autophagy, thereby promoting tumor cell death.

Conclusions

Our study defines novel molecular mechanisms underpinning the antitumor actions of P2X7 and provides a further rationale for purine-based drugs in targeted cancer therapy.     

Noninvasive Assessment of Response to Neoadjuvant Chemotherapy in Osteosarcoma of Long Bones with Diffusion-Weighted Imaging: An Initial In Vivo Study

Objectives

The purpose of our study is to investigate whether diffusion-weighted imaging (DWI) is useful for monitoring the therapeutic response after neoadjuvant chemotherapy in osteosarcoma of long bones.

Materials and methods

Conventional magnetic resonance imaging (MRI) and DWI were obtained from 35 patients with histologically proven osteosarcomas. MR examinations were performed in all patients before and after 4 courses of preoperative neoadjuvant chemotherapy. Apparent diffusion coefficients (ADC) were measured. The degree of tumor necrosis was assessed macroscopically and histologically by two experienced pathologists after operation. Student’s t test was performed for testing changes in ADC value. Pearson’s correlation coefficient was used to estimate the correlation between necrosis rate and post- neoadjuvant chemotherapy ADC values. P<0.05 was considered to denote a significant difference.

Results

The difference of the whole osteosarcoma between pre- neoadjuvant chemotherapy ADC value (1.24±0.17×10⁻³ mm²/s) and post- (1.93±0.39×10⁻³ mm²/s) was significant difference (P<0.01). Regarding in patients with good response, the post- neoadjuvant chemotherapy values were significantly higher than the pre- neoadjuvant chemotherapy values (P<0.01). The post- neoadjuvant chemotherapy ADC value in patients with good response was higher than that of poor response (t = 8.995, P<0.01). The differences in post- neoadjuvant chemotherapy ADC between viable (1.03±0.17×10⁻³ mm²/s) and necrotic (2.38±0.25×10⁻³ mm²/s) tumor was highly significant (t = 23.905, P<0.01). A positive correlation between necrosis rates and the whole tumor ADC values (r = 0.769, P<0.01) was noted, but necrosis rates were not correlated with the ADC values of necrotic (r = −0.191, P = 0.272) and viable tumor areas (r = 0.292, P = 0.089).

Conclusions

DWI can identify residual viable tumor tissues and tumor necrosis induced by neoadjuvant chemotherapy in osteosarcoma. The ADC value can directly reflect the degree of tumor necrosis, and it is useful to evaluate the preoperative neoadjuvant chemotherapy response in patients with osteosarcoma.     

Dual‐function fluorescent probe for cancer imaging and therapy

To date, several fluorescent probes modified by a single targeting agent have been explored. However, studies on the preparation of dual‐function quantum dot (QD) fluorescent probes with dual‐targeting action and a therapeutic effect are rare. Here, a dual‐targeting CdTe/CdS QD fluorescent probe with a bovine serum albumin–glycyrrhetinic acid conjugate and arginine‐glycine‐aspartic acid was successfully prepared that could induce the apoptosis of liver cancer cells and showed enhanced targeting in in vitro cell imaging. Therefore, the as‐prepared fluorescent probe in this work is an efficient diagnostic tool for the simultaneous detection of liver cancer and breast cancer cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Stage 2 Process Performance Qualification (PPQ): a Scientific Approach to Determine the Number of PPQ Batches

The approach documented in this article reviews data from earlier process validation lifecycle stages with a described statistical model to provide the “best estimate” on the number of process performance qualification (PPQ) batches that should generate sufficient information to make a scientific and risk-based decision on product robustness. This approach is based upon estimation of a statistical confidence from the current product knowledge (Stage 1), historical variability for similar products/processes (batch-to-batch), and label claim specifications such as strength. The analysis is to determine the confidence level with the measurements of the product quality attributes and to compare them with the specifications. The projected minimum number of PPQ batches required will vary depending on the product, process understanding, and attributes, which are critical input parameters for the current statistical model. This new approach considers the critical finished product CQAs (assay, dissolution, and content uniformity), primarily because assay/content uniformity and dissolution as well as strength are the components of the label claim. The key CQAs determine the number of PPQ batches. This approach will ensure that sufficient scientific data is generated to demonstrate process robustness as desired by the 2011 FDA guidance.     

High glucose concentration induces endothelial cell proliferation by regulating cyclin‐D2‐related miR‐98

Cyclin D2 is involved in the pathology of vascular complications of type 2 diabetes mellitus (T2DM). This study investigated the role of cyclin‐D2‐regulated miRNAs in endothelial cell proliferation of T2DM. Results showed that higher glucose concentration (4.5 g/l) significantly promoted the proliferation of rat aortic endothelial cells (RAOECs), and significantly increased the expression of cyclin D2 and phosphorylation of retinoblastoma 1 (p‐RB1) in RAOECs compared with those under low glucose concentration. The cyclin D2‐3′ untranslated region is targeted by miR‐98, as demonstrated by miRNA analysis software. Western blot also confirmed that cyclin D2 and p‐RB1 expression was regulated by miR‐98. The results indicated that miR‐98 treatment can induce RAOEC apoptosis. The suppression of RAOEC growth by miR‐98 might be related to regulation of Bcl‐2, Bax and Caspase 9 expression. Furthermore, the expression levels of miR‐98 decreased in 4.5 g/l glucose‐treated cells compared with those treated by low glucose concentration. Similarly, the expression of miR‐98 significantly decreased in aortas of established streptozotocin (STZ)‐induced diabetic rat model compared with that in control rats; but cyclin D2 and p‐RB1 levels remarkably increased in aortas of STZ‐induced diabetic rats compared with those in healthy control rats. In conclusion, this study demonstrated that high glucose concentration induces cyclin D2 up‐regulation and miR‐98 down‐regulation in the RAOECs. By regulating cyclin D2, miR‐98 can inhibit human endothelial cell growth, thereby providing novel therapeutic targets for vascular complication of T2DM.