i-Fect Scores Again

Knock-down of HIF-1a Attenuates Chemo Induced Pain
i-Fect ^TM is one of our original products. It has enjoyed 14 years of upping transfection percentages both in-vitro and in-vivo.

Here is a new study showing successful use of i-Fect to knock down HIF-1a in-vivo-Taylor Ludman and Ohannes K. Melemedjian. (2019). Bortezomib-induced aerobic glycolysis contributes to chemotherapy-induced painful peripheral neuropathy. Molecular Pain. https://doi.org/10.1177/1744806919837429.

Figure 1. (a) Treatment of mice with bortezomib (Bor) for five days augmented HIF1A expression in L4-6 DRGs (*P ¼ 0.0412, five mice/ group) relative to the vehicle-treated group. (b) A schematic depicting the site of the intrathecal (IT) siRNA injection. The siRNA was administered between the L4 and L5 vertebrae which is around 17 mm rostral to the spinal cord (SC) section innervated by the L4-6 DRGs. (c) IT injection of siRNA (1 mg in 5 ml) that targets HIF1A (siRNA) but not control siRNA (Cont), for two consecutive days, significantly reduced the levels of HIF1A in L4-6 DRGs. (***P=0.0006, five mice/group). (d) IT siRNA did not affect HIF1A levels in L4-6 spinal cord (five mice/group). (e) After determining baseline withdrawal thresholds using von Frey filaments, male ICR mice received IP injection of vehicle or bortezomib (black arrows) and IT siRNA (blue arrows). The withdrawal thresholds were measured on days 7 to 14. IT HIF1A siRNA prevented the development of bortezomib-induced neuropathic pain. (****P less than 0.0001, five mice/group). DRG: dorsal rootganglia; HIF1A: hypoxia-inducible factor 1 alpha; IT: intrathecal; IP: intraperitoneal; siRNA: small interfering RNA.

This study is the first to demonstrate that the stabilization of HIF1A expression underpins the development of bortezomib-induced neuropathic pain. Crucially, these findings reveal that the initiation and maintenance of bortezomib-induced neuropathic pain are regulated by distinct mechanisms.

Magic Red and Cancer Research

Measure Apoptosis in Real Time in Cancer Cells

Magic Red™ (MR) Kits measure apoptosis via active caspases and cathepsins in whole living, intact cells - no lysis required. As apoptosis progresses and caspase activity increases, the red fluorescent signal increases.

In this example, Magic Red is used to measure apoptosis in HeLa Cells after treatment with Artesunate (ART), an anti-malarial drug and potential chemotherapy agent.

Figure: ART activates lysosomal function. A, HeLa cells were first treated with ART (50 μM) for 12 h, followed by LTR labeling for 30 min, and further analyzed using confocal microscopy. B, HeLa cells were loaded with LTR following time course drug treatment, and fluorescence intensity of 10,000 cells/sample was measured by flow cytometry. C, HeLa cells were treated as indicated in A and then incubated with Magic Red cathepsin L and LTG for 30 min and observed under a confocal microscope. D, HepG2 cells were treated as in A and then labeled with LTR described earlier in A. E, HepG2 cells were treated as in A, followed by a Magic Red cathepsin B and L assay for 45 min. Fluorescence intensity of 10,000 cells/sample was measured using flow cytometry. F, HeLa cells were first loaded with DQ Red BSA for 1 h and then treated with ART (50 μM) for 18 h and incubated with LTG for 30 min. G, HeLa cells were treated with ART for 18 h and then immunostained for LAMP1. H, LAMP1 signals of 80 cells from three independent experiments were quantified by ImageJ (Student's t test, #, no significance). I, HeLa cells were treated with ART for the indicated time, and then Western blot was performed to detect the LAMP1 protein level. Scale bar, 10 μm. Error bars, S.D. doi: 10.1074/jbc.M114.564567

We have a variety of Apoptosis Kits for testing potential Chemotherapeutic Agents.

Extracellular Matrix Environment and Chemotherapeutics

Substrate Matters!

Neuromics' has been promoting a variety of 3-D Cell Based Assay Solutions for the past several years. These include: Nanofibers, Hydrogels and Extracellular Matrix (ECM) Proteins. We have found that the adoption rate for these as standard tools for drug discovery is slower that we anticipated.

We believe substrate matters so I am pleased to share a recent publication that references use of our Collagen IV and other ECM proteins. This confirms the importance of using a more in vivo like environment in testing chemotherapeutics: Thuy V. Nguyen,Marianne Sleiman,Timothy Moriarty,William G. Herrick,Shelly R. Peyton. Sorafenib resistance and JNK signaling in carcinoma during extracellular matrix stiffening. Publication: Biomaterials. Elsevier. 13 April 2014. http://dx.doi.org/10.1016/j.biomaterials.2014.03.058.

Abstract: Tumor progression is coincident with mechanochemical changes in the extracellular matrix (ECM). We hypothesized that tumor stroma stiffening, alongside a shift in the ECM composition from a basement membrane-like microenvironment toward a dense network of collagen-rich fibers during tumorigenesis, confers resistance to otherwise powerful chemotherapeutics. To test this hypothesis, we created a high-throughput drug screening platform based on our poly(ethylene glycol)-phosphorylcholine (PEG-PC) hydrogel system, and customized it to capture the stiffness and integrin-binding profile of in vivo tumors. We report that the efficacy of a Raf kinase inhibitor, sorafenib, is reduced on stiff, collagen-rich microenvironments, independent of ROCK activity. Instead, sustained activation of JNK mediated this resistance, and combining a JNK inhibitor with sorafenib eliminated stiffness-mediated resistance in triple negative breast cancer cells. Surprisingly, neither ERK nor p38 appears to mediate sorafenib resistance, and instead, either ERK or p38 inhibition rescued sorafenib resistance during JNK inhibition, suggesting negative crosstalk between these signaling pathways on stiff, collagen-rich environments. Overall, we discovered that β1 integrin and its downstream effector JNK mediate sorafenib resistance during tumor stiffening. These results also highlight the need for more advanced cell culture platforms, such as our high-throughput PEG-PC system, with which to screen chemotherapeutics.

Figure: High-throughput biomaterial platform for drug screening. (A) The high-throughput platform consists of a black-walled, glass bottom plate, with PEG-PC gels cast in each of the inner 6x10 wells. (B) Gels can be functionalized with any protein or peptide of interest, and they support the adhesion and growth of carcinoma cells. We used this platform to test carcinoma cell response to a kinase inhibitor (sorafenib) as a function of underlying gel stiffness and ECM adhesive protein cocktail. (C) A representative graph of SkBr3 proliferation (y-axis) in response to sorafenib (x-axis) across a range of gel stiffness (colors) demonstrates the IC-50 calculation.

This confirms the importance of considering your substrate environment when developing your in vitro assays for High Content and High Throughput Drug Discovery. We will continue to provide updates.

Cannabinoid Type-1 Receptors and Chemotherapy Related Pain

Chemotherapy can induce painful peripheral neuropathy and also have a toxic effect on peripheral nerves. The authors here show that that cisplatin produces hyperalgesia and toxicity to sensory neurons as indicated by neurochemical, morphological, and functional measures. Increasing AEA signaling at CB1 receptors not only reduced the hyperalgesia but reduced the neurotoxicity of cisplatin as well:  Iryna A. Khasabova,Sergey Khasabov, Justin Paz, Catherine Harding-Rose, Donald A. Simone, and Virginia S. Seybold. Cannabinoid Type-1 Receptor Reduces Pain and Neurotoxicity Produced by Chemotherapy. The Journal of Neuroscience, 16 May 2012, 32(20): 7091-7101; doi: 10.1523/​JNEUROSCI.0403-12.2012.

The authors use our guinea pig TRPV1 antibody to measure cisplatin hyperalgesia vs treated and control mice.

Images: URB597 attenuated effects of cisplatin on protein expression in DRGs. A, TRPV1- and ATF-3-ir were detected by immunofluorescence in L3–L5 DRGs from mice treated with vehicle, cisplatin, or cisplatin plus URB597. Cisplatin (1 mg/kg of body weight, daily for 7 d, i.p.) increased the occurrence of TRPV1- and ATF3-ir in neurons. Co-injection of URB597 (0.3 mg/kg daily, i.p.) with cisplatin attenuated the effect cisplatin on protein-ir. Scale bars: 10 μm (for images within each antigen). B, Quantitative summary of the effect of treatments on TRPV1-ir in neurons. Data are expressed as the mean ± SEM. aSignificantly different from each other group (p < 0.05, one-way ANOVA with Student–Newman–Keuls test; n = 4 mice/treatment). C, Quantitative summary of the effect of treatments on ATF-ir in neurons. Data are expressed as the median and 25th and 75th percentile range. *Significantly different from vehicle control and cisplatin plus URB597 groups (n = 6 mice/treatment; p < 0.001, Kruskal–Wallis ANOVA on ranks test).

Related Reagents:

VR1 N-Terminus (TRPV1)
VR1 (TRPV1)-Goat
VR1 C-Terminus (TRPV1) - mouse specific
All TRP Antibodies
Pain and Inflammation Research Antibodies
Neurotransmission -Neurotransmission Research Antibody Categories