Immunohistochemical staining of paraffin-embedded human lung cancer using Proteintech’s KRAS antibody (cat no. 12063-1-AP, viewed under 40x lens).

Lung cancer remains a leading cause of cancer-related death worldwide due to its oft advanced stage at discovery, in combination with its difficult-to-treat and complex nature. Non-small cell lung cancer (NSCLC), for example, classifies some 85 per cent of lung cancer cases, and tumors in this category can harbor mutations in any of several genes including: KRAS, EGFR, BRAF, ALK, RET and TP53. Platinum-based combination chemotherapy is currently the first-line of treatment for NSCLC; however, the prognosis for advanced-stage patients remains poor ― new therapies are urgently needed.

Blockade of programmed cell death 1 ligand 1 (PD-L1) binding to its receptor programmed cell death 1 (PD-1) is a promising immunotherapeutic approach in cancer medicine. Several types of cancer, including NSCLC, respond to treatment with nivolumab [1] ― a therapeutic antibody that blocks PD-1 binding to PD-L1, an important checkpoint in the adaptive immune response.

The PD-1–PD-L1 interaction is normally employed to switch T-cells off (for example when  T-cell activation has peaked in an immune response to prevent host tissue damage); however, PD-L1 expression can be altered and exploited by cancer in an attempt to evade the immune system. Though it is evolved to eliminate cancer quite effectively, scientists now understand that the adaptive immune response can be suppressed and held at bay by the display of immune checkpoint ligands, such as PD-L1, on tumors (almost like molecular camouflage).

Separate NSCLC cell lines carrying KRAS, EGFR, BRAF, ALK or RET mutations have been found to express high levels of PD-L1, and this may be linked to high levels of PI3K/Akt/mTOR pathway activation according to work presented at the last AACR Annual Meeting [2].The authors, based at Johns Hopkins University, found concurrently high levels of active Akt and PD‑L1 expression in lung cancer-derived cell lines. Furthermore, manipulation of the PI3K/Akt/mTOR pathway in these cells also affected PD-L1 expression accordingly. Ultimately, the work demonstrated that combining PD‑1/PD-L1 pathway blockade with inhibition of the PI3K/Akt/mTOR signaling pathway (in this case, using the mTOR inhibitor rapamycin), enhanced tumor shrinkage in mice bearing lung cancer xenografts. This may provide a valuable drug combination strategy for the treatment of NSCLCs proving to be more immunotherapy-resistant.

An additional approach to compliment PD‑1/PD-L1 pathway blockade was also showcased at the AACR this year, this time involving epigenetic manipulation [3]. John Wrangle (MD, MPH), a Young Investigator on the Stand Up to Cancer (SU2C) Epigenetics Dream Team, delivered a talk describing the cumulative effects of epigenetic therapy immediately followed by PD‑1/PD-L1 pathway blockade. Successful outcomes were seen in five out of five patients in this small-scale study, where patients received the histone deacetylase (HDAC) inhibitor entinostat and the demetylating agent azacitidine (Aza) in combination prior to PD‑1/PD-L1 pathway blockade.

In further investigations it was discovered that even low doses of Aza had diverse immunomodulatory effects in eight separate NSCLC cell lines – which included up-reglation of the Jun/Jnk, NFkB, antigen processing and antigen presentation pathways. Wrangle and the SU2C Epigenetics Team suspect that Aza elicits these effects via inhibition of DNA methyltransferase proteins, with DNMT1 and DNMT3b being prime candidates. Based on these findings, the team is currently developing a clinical trial that will test the efficacy of DNMT and HDAC inhibition combined with blockade of the PD-1/PD-L1 pathway in cancer patients. Hopefully, the successes of the initial five patient trial can be echoed in this one, giving patients more treatment options and survival hope.

[Author: Deb Grainger]

Related Antibodies

Akt antibodies

EGFR-specific antibody (catalog no. 18986-1-AP)

HDAC antibodies

Jnk rabbit polyclonal antibody (catalog no. 10023-1-AP)

Jun rabbit polyclonal antibody (catalog no. 10024-1-AP)

KRAS antibodies

NFkB p65 rabbit polyclonal antibody (catalog no. 10745-1-AP)

p53 rabbit polyclonal antibody (catalog no. 10442-1-AP) 

PD-1 rabbit polyclonal antibody (catalog no. 18106-1-AP)

PD-L1 rabbit polyclonal antibody (catalog no. 17592-1-AP)

PI3K antibodies

References

[1] Topalian S L et al., N Engl J Med 2012; 366:2443-2454

[2] Lastwika K et al., PI3K/AKT/mTOR pathway activation drives expression of the immune inhibitory ligand PD-L1 in NSCLC, abstract no. 4981, poster presentation at the American Association for Cancer Research Annual Meeting 2013, April 6-10, Washington DC.

[3] Wrangle J et al., Epigenetic therapy and sensitization of lung cancer to immunotherapy, abstract no.  4619, talk presentation at the American Association for Cancer Research Annual Meeting 2013, April 6-10, Washington DC.

The post PD-L1 and Lung Cancer Research: Pathways to Promising Immunotherapies appeared first on The Proteintech Blog.

Scanning electron micrograph of a human T-cell (Source: Wikimedia Commons, Original Source: NIH/NIAID.)

The immune system’s role in cancer was a recurring topic at this year’s American Association of Cancer Research (AACR) Annual Meeting. From the outset of the conference, talks and poster presentations detailing how the immune response can be primed to combat cancer seemed ubiquitous; a reflection of the progress made in the field of cancer immunology over the past decade.

Sunday’s plenary speaker Suzanne Topalian, Professor of Surgery and Oncology at Johns Hopkins University, explained how a shift in the way scientists view the immune system and cancer has contributed to advances in her field: “We’ve come to realize that the overriding relationship between the immune system and cancer is one of tolerance, rather than rejection.”

This view has changed the trajectory of immunological medicine somewhat; now the questions being asked are along the lines of ‘How can the immune system be started up again to fight cancer?’ Or how do we, as Topalian put it, “Release the brakes on the immune system.”

One strategy cancer cells use to dupe the immune system into applying the brakes is to display signaling molecules on their surface that inhibit T-cells. This mimics a normal process in the body which helps the adaptive immune system recognize and prevent destruction of ‘self’ after initiation of an immune response; effectively, it switches T-cells off. Using this mechanism to their advantage, cancer cells go unchecked by the immune system – but scientists have been aware of this tactic for some time and have developed ways to counteract it.

One of the signaling molecules used as molecular ‘camouflage’ by cancer in immune system evasion is programmed cell death 1 ligand 1 (PD-L1, also known as B7-H1), the predominant ligand of the programmed death 1 (PD-1) receptor. It was shown that many human cancers display PD-L1 on their surface , whereas most normal human tissues do not [1]. Moreover, preclinical work shows that PD-L1 binding to its receptor has a pivotal role in local cancer immunosuppression as well as in mobilizing the immune system against cancer [2].

Clinically, PD‑1 receptor blockade might be a lifeline for those patients whose diseases are harder to treat. Topalian and colleagues designed a study in which patients were required to have undergone at least one round of unsuccessful treatment with another form of therapy prior to recruitment – some of the patients had received up to five previous failed treatments. Five different types of cancer were represented in the study, including non-small cell lung cancer (NSCLC)  a cancer that is historically resistant to immunotherapy.

Despite this bias towards treatment-resistant disease, a measurable response to the PD‑1 blockade therapy was seen in approximately one in four to one in five patients in the trials [3]. Success was even seen in cases of intermittent therapy. However, one of the most promising outcomes of the nivolumab studies arose from follow‑up work carried out more than three years after cessation of the trial therapy [4]. Patients who responded successfully to the initial nivolumab treatments were invited back for assessment.  The outcomes for these patients demonstrated maintained disease improvement with the PD‑1 targeted therapy long after treatment ended in some cases – one such patient still showed a complete response ‘off-drug’ five years later.  Metastatic relapse could even be reversed with reinduction of nivolumab therapy. Of these observations Topalian said: “These results are compatible with the knowledge that the immune system [remembers], that this is actually a living therapy that stays with the host, potentially for many years.”

Back on the lab bench, PD-L1 is currently being assessed as a biomarker for PD-1 blockade treatment efficacy. The ligand is already being employed as a qualifier for patient entry onto certain clinical trials. This is not the approach Topalian prefers, stating: “We could learn more by continuing to treat the total population of patients and follow the biomarker’s [expression].”   This way Topalian hopes to identify types of cancer that should be tested routinely for PD-L1 positivity.

[Author: Deb Grainger]

Related Antibodies

Anti-PD-L1 rabbit polyclonal antibody (catalog no. 17592-1-AP)

Anti-PD-1 rabbit polyclonal antibody (catalog no. 18106-1-AP)

References

[1] Dong H et al., Nature Medicine 2002;8:793-800

[2] Currie A J et al., J Immunol. 2009;183(12):7898-908

[3] Topalian S L et al., N Engl J Med 2012; 366:2443-2454

[4] Lipson E J et al. Clin Cancer Res. 2013;19(2):462-8

The post PD-1 Blockade: Priming the Immune System to Combat Cancer appeared first on The Proteintech Blog.

Tiago Outeiro, Professor of Neurodegeneration at  the University of Goettingen Medical School.

Currently, neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s are the focus of Tiago Outeiro’s lab. I ask what led him to study such a huge scope of neurodegeneration, where most focus on just one disease; he explains that the common thread linking these diseases together is his main concern…

“I became interested in the field of neurodegeneration in the 90s when people were still talking about ‘mad cow disease’.”  He begins, using the lay term for the disease at the heart of the bovine spongiform encephalopathy (BSE) scare of the 80s and 90s, which often umbrellas the human Creutzfeldt jakob diseases (CJDs) too — though only variant CJD is passed from cow to human.

“At that time the disease mechanisms of BSE and CJD were not very clear; it was known that they were neurodegenerative, characterized by the loss of certain types of brain cells, and related to protein aggregation. It was very intriguing to find that the ‘infectious’ agent causing these types of diseases was in fact a protein.” A nod towards the still perplexing subject of prion proteins, which are infectious and destructive when misfolded, but seemingly vital for cellular function when intact.

He explained the significance of this: “It was a completely new agent; it was not a bacteria or a virus – but it was infectious and transmissible. So, as a graduate student, I was very interested in the study of prions. From there things just evolved and I started studying other proteins that would also misfold and aggregate. I began working on Parkinson’s and Alzheimer’s and Huntington’s disease – that’s how it started.”

Though the protein suspects in each disease line-up may differ, they seem to have aggregation — and now propagation — mechanisms in common. So what is the current understanding of the roles such mechanisms play in neurodegenerative diseases? Outeiro describes how aggregation in particular might not be contributing to neurodegeneration as one would think…

“The majority of the diseases I study have been acknowledged for some time, some of them for more than a hundred years. One of the earliest pathological observations doctors and researchers made in studying them was the presence of protein inclusions — clumps of aggregated protein — sitting either inside or just outside neurons in diseased brains. At the time people said, ‘Okay, these big protein clumps must be doing something very bad to the cells — they are the root of the disease.’” And as the science textbooks show, the main thrust of neurodegeneration research ran with this hypothesis.

“Over time, a lot of research has brought us the current view that maybe the protein aggregates, the big clumps that we see, are not actually the disease perpetrators — aggregation may be a protective mechanism the cell has evolved in an attempt to get rid of toxic protein species.” These toxic species are the things causing damage to the neurons and, as Outeiro explains, are probably much smaller, invisible to the pathologist. He describes how this might work in simple terms:

“Imagine that you have a lot of trash spread out in a room, it creates a lot more clutter than if you just put the trash all together in one corner. Even if the pile gets bigger, it doesn’t create as much disruption as leaving it around the room.” Presumably, where neurodegenerative disease develops is when this defense system becomes overloaded.

I ask Tiago where he thinks understanding neurodegenerative disease in this way will take us in terms of new discoveries in treatment and prevention.

“Understanding the molecular mechanisms behind every facet — protein misfolding, aggregation, how the cells clear up these protein aggregates, which pathways are involved — will, hopefully, identify new targets we can use for therapeutic intervention. The hope is that we can develop small molecules and inhibitors (if the targets are enzymatically active) that  modulate relevant cellular pathways to help them clear the toxic species more effectively and, ultimately, benefit patients.”

Data from the Tiago Outeiro lab

A potential caveat presents itself here: if you target and modulate the “trash collecting” mechanism, could this interfere with the body’s natural defense systems against toxic proteins in an undesirable way?

“At the moment this is also a big question; if we tweak pathways that normally function as in a certain way to do things differently, then of course this could create other problems that we do not foresee right now. That is why it’s very important to understand all aspects of the problem, to get to its root ― uncover the very basic molecular mechanism.

To do this, Outeiro’s group has even employed yeast to help them model certain aspects of neurodegenerative diseases.

“The way you normally study these diseases is to use a mouse model ― all agree that this is closer to humans than yeast. Yet yeast still have the advantage of being eukaryotic cells as well; they share a lot of similarities, in terms of their basic functioning, with mammalian cells ― often with human cells in particular. This presents the possibility of doing certain types of studies and genetic screens that cannot be done in other systems.”

“Of course we cannot say we are going to be curing any of these human diseases just by using yeast as a model. But we can certainly explore all the potential of yeast genetics and tools that have been developed over the years to try to understand what is happening at a very basic level, in a very detailed way. We can then test those findings in other, more complex models; if everything still works the way we think, then we’ve made some progress.”

A newer technique being refined in the Outeiro lab might help them make vital progress that much quicker; it enables them to visualize and study protein misfolding and aggregation in the living brain. He explains the reasoning behind developing the technology…

“The limitation in most studies is that we look at still images of the brain, and therefore miss the dynamics of the different disease processes. If we can image the process of protein aggregation in the living brain we will be much closer to understanding how things develop.”

This promising technology certainly sounds hi-tech: “For the [live brain] imaging we use 2-photon microscopy, a technique that uses a laser that does no damage to the brain tissue yet enables us to see protein dynamics in at least the outer layers of the cortex, through what we call a cranial window. This avoids the need to sacrifice animals (small rodents like mice or rats) in order to observe and interfere with protein aggregation.”

So could technology like this ‘clear up’ our understanding of the role of protein aggregation in disease? It is definitely one of Outeiro’s goals. But, I’m curious, could it also be used to detect neurodegenerative disease? Well the answer is sort of yes and no; it could help with earlier detection, but in a more roundabout way than I imagined ― still, improving disease detection is still on Outeiro’s to-do list…

“Indeed, if we can understand all the players involved in a certain neurodegenerative disease we might be able to detect the real culprit of it earlier. A deeper understanding of the molecular mechanisms involved is essential for the development of novel diagnostic test; thus, we hope these studies will bring us closer to this goal as well.”

The German Neuroscience Society National Meeting

The GNS National meeting is held every two years in Goettingen, Germany. (Source: Wikimedia Commons)

I spoke to Professor Outeiro at the last installment of the GNS national meeting, for which he organized and chaired a session entitled “Molecular mechanisms and spreading of alpha-Synuclein pathology in the brain”. He said says of the meeting and the success of his session:

“The GNS meeting is very important for me in particular because I only moved to the University of Goettingen recently. It is a good way for me to get acquainted with my other colleagues in Germany and the research going on in their labs. We’re lucky in Goettingen because of our cental location and the GNS meeting is usually organized here. It attracts more than 2,000 participants, which provides a great opportunity to meet colleagues, learn about their work, and establish future collaborations here in Germany ― So, it’s very good for that!

“A very important and very hot area at the moment is trying to understand how alpha-synuclein spreads in the brain, because this could have a huge implications for our understanding of how Parkinson’s disease  progresses. At this point I thought it would make sense to have a symposium, and invite experts in the field, to inform the community here in Germany of what is being done in alpha-synuclein research and the important things yet to investigate therein.

“The session itself went well, we had great science being presented. For instance we had a graduate student presenting her work on the LRRK2 protein, which is a new subject in this area as it is usually regarded as independent of alpha-synuclein research. However, we now understand that there is a strong connection between the two, and finding an alpha-synuclein interaction with LRRK2 is again bringing us back to the question of protein aggregation and how the cells process these two proteins.”

Author: Deborah Grainger

Related antibodies

Anti-alpha synuclein (SNCA) polyclonal antibody (catalog no. 10842-1-AP) 

Anti-APP polyclonal antibody (catalog no. 10524-1-AP)

The post Protein Aggregation: Culprit or Custodian in Neurodegeneration? | Interview with Tiago Outeiro appeared first on The Proteintech Blog.

DNA Repair is a process vital to all mammalian life forms (shown here: DNA Ligase 1. Source: Wikimedia Commons.)

Guest post by Katie Griffiths

Base excision repair is a crucial mechanism involved in DNA repair and protection. The process is initiated by DNA glycosylases, which recognize and cleave damaged or incorrectly paired bases. Owing to the importance of this process, considerable study has gone into understanding the cellular and molecular mechanisms involved, and the ways in which these mechanisms are regulated.

Human NEIL3 is one of a trio of DNA glycosylases involved in base excision repair [1]. Previously, it was confirmed that both NEIL1 and NEIL2 proteins are active DNA glycosylases, but questions surrounding NEIL3’s activity remained. Recently, Proteintech’s anti-NEIL3 antibody was utilized in research undertaken at the University of Oslo demonstrating the DNA glycosylase activity of endogenous human NEIL3 for the first time [2]. Establishing this activity of NEIL3 had been a significant challenge as the full-length recombinant enzyme proved difficult to purify. However, in this novel study the authors Neurauter et al. were able to obtain active NEIL3 from two human cell lines using the Proteintech anti-NEIL3 antibody for immunoprecipitation.

Next, in order to probe the specific role of the NEIL3 protein in cell turnover, the Oslo group used the same anti-NEIL3 antibody to determine the expression levels of the protein during cell cycle progression. They synchronized cell division in cell populations using serum deprivation and cell-cell contact inhibition — essentially pausing the cell cycle and inducing quiescence. After re-initiation of the cell cycle, the authors monitored the levels of all three NEIL proteins during its different phases. The results demonstrated that NEIL3 is expressed at distinct times during the cell cycle, being tightly regulated during quiescence but quickly induced and highly expressed during S phase through to G2 phase, the later stages of the cycle. Moreover, the authors also discovered the molecules regulating NEIL3 differ from that of NEIL1 and NEIL2; expression of NEIL3 was noted to be at least partially dependent on activation of the ERK/MAP kinase pathway.  

The expression pattern of NEIL3, including its suppression during quiescence and induction during S phase, are strong indication that it plays a role in base excision repair specifically during replication. This is supported by a previous study carried out by the same research group, identifying colocalization of NEIL3 with replication protein A [3] — an essential protein in DNA replication preventing single-stranded DNA from winding back on itself.

If NEIL3 is indeed a key component of replication-specific base excision repair, it is of potential consequence to the field of cancer research as a very large proportion of human tumors demonstrate regulation problems at the S/G1 transition phase. Furthermore upregulation of NEIL3 expression has been demonstrated in several cancers including melanoma [4]. Further study into the expression of NEIL3, and particularly its regulation by the ERK/MAPK pathway, will provide greater insight into its potential role in cancer biology.

Another significant area of NEIL3-based research is in the field of neurogenesis; previous to confirming the glycosylase activity of NEIL3, the University of Oslo researchers also discovered a potential specific role for this protein in the developing brain. Their results showed that mouse Neil3 was not uniformly expressed in the brain, but limited to specific regions known to harbor neural stem and progenitor cells. The expression appeared to be tightly regulated both spatially and temporally during brain development [5].

Guest Blogger profile

Katie Griffiths is a freelance scientific editor, technical writer and science communicator. She has recently graduated with a Research Masters in Structural Biology from The Institute of Structural and Molecular Biology at Birkbeck College. In her spare time she writes a blog The Molecular Circus, enthusing her readers about the molecular biology of everyday life.

Related antibodies

NEIL3 rabbit polyclonal antibody (catalog no. 11621-1-AP)

NEIL1 rabbit polyclonal antibody (catalog no. 12145-1-AP)

LIG4 rabbit polyclonal antibody (catalog no. 12695-1-AP)

LIG1 rabbit polyclonal antibody (catalog no. 18051-1-AP)

References

[1] Bandaru V et al., DNA Repair (Amst). 2002;1(7):517-29 

[2] Neurauter CG et al., DNA Repair (Amst). 2012;11(4):401-9

[3] Morland I et al., Nucleic Acids Res. 2002;30(22):4926-36

[4] Kauffmann A et al., Oncogene. 2008;27(5):565-73 

[5] Hildrestrand GA et al., BMC Neurosci. 2009;10:45

The post NEIL3 Antibody Used to Investigate Mechanisms of DNA Repair appeared first on The Proteintech Blog.

Saturday

A mammographic image to detect breast cancer. Source: Wikimedia Commons via University of Copehnage University Post.

The official start of the meeting on Saturday sees a wealth of Educational Sessions and Methods Workshops taking place. Of these, the Educational Session “Deconstructing the Complexity of Triple Negative Breast Cancer” features on my list. Cancer heterogeneity has become a big focus area, especially in light of such findings that there may be at least ten subtypes of breast cancer. This has altered the thinking that the majority of breast tumors fall into any of three main groups (HER2+, ER+ and PR+*) with the remaining 15 per cent being classed under ‘other’. Members of this ‘other’ group are officially known  as triple-negative breast cancer ― a group comprised of invasive, aggressive breast tumors, lacking canonical markers.  The “Deconstructing the Complexity of Triple Negative Breast Cancers” session seeks to discuss the breakdown of triple-negative tumors into further subtypes, distinguishable by newly uncovered features, such as distinct molecular, histopathological and drug-response profiles.

Session chair Jorge Reis-Filho and presenter Nicholas Turner have both appeared as senior authors on separate papers investigating unique aspects of triple-negative breast cancers, works that utilized Proteintech’s anti-PLEKHF1 [1] and anti-GMNN [2] antibodies respectively.

Sunday

The secondary structure of a precursor microRNA sequence from Brassica oleracea, as predicted by MFOLD. Source Wikimedia Commons, Authors: Stewart and M. Zuker (2007).

MicroRNAs or noncoding RNAs (ncRNAs) have been a hot topic of the biological world for at least a decade since their functional role in gene expression and regulation was established. Several extended families of ncRNAs have since been characterized and together they are known to control the expression of approximately half of all genes in mammals – a well-known example of gene regulation by ncRNAs is X-chromosome inactivation, coordinated by the long ncRNA XIST. In light of this role in gene regulation, it is not surprising then that many ncRNAs show abnormal expression patterns in cancerous tissues. The “Noncoding RNAs and Cancer” major symposium in Ballroom C on Sunday will present the latest work in the area of ncRNAs and their roles in malignancy.

 

Despite the RNA-focus of the talks in this session, Proteintech antibodies might also appear: speaker Joshua Mendell has co-authored a paper featuring Proteintech’s anti-DGCR8 antibody, entitled “Cell-cell contact globally activates microRNA biogenesis” [3].

Monday

There are a few sessions on Monday that have caught my eye, but (as is the norm with meetings of this size) there are timetable clashes. Therefore, I may attempt to view individual talks of interest in each of these sessions (geographical locations allowing!) First up is Domagoj Vucic’s talk “Targeting IAP proteins for the treatment of cancer” in the “Expanding the Druggable Genome” session starting at 1 pm. Vucic’s talk is the first of the session, running till 1:30 pm, and will present the inhibitor of apoptosis (IAP) protein family as possible drug targets for overcoming cancer resistance to treatment and evasion of cell death.

Vucic, a scientist at Genentech, demonstrated previously that both c-IAP1 and c-IAP2 are critical mediators of TNFα-induced NF-κB activation, and that combined absence of c-IAP1 and c-IAP2 renders cells sensitive to TNFα-induced cell death. This work also made use of Proteintech’s anti-c-IAP1 antibody for detection of this target by Western blotting [4].

I’ve got my fingers crossed that the five minute gap between Vucic’s talk  in room 202 and John Carpten’s talk in the “Use  of Next Generation Sequencing: Implementation of Clinical Genomics” major symposium in Ballroom C is enough time to move between the two. Carpten will be presenting his talk “Genome sequencing as a nexus for cancer therapy” in theme with the main topic of the symposium: personalized medicine – a major trajectory of modern cancer therapy.

Immunofluorescence staining of HepG2 cells using Proteintech’s DDX21 antibody (10528-1-AP, 1:50 dilution) and rhodamine-labeled goat anti-rabbit IgG (red). Blue staining = DAPI stained nucleus.

Carpten’s talk may touch on work undertaken with colleagues at the TGEN institute, identifying a somatic mutation in the AKT1 gene which appears almost exclusively in breast cancers of luminal origin [5]. A second paper that describes the effects of the resulting glutamic acid to lysine substitution in the AKT1 protein also features Proteintech’s anti-DDX21 antibody [6], which has also appeared in several other cancer research papers.

The tight schedule of Monday afternoon means another five-minute dash to room 151 for Elaine Fuchs’ talk at 2 pm: “Skin stem cells in morphogenesis in cancer”. Proteintech has some great antibodies against stem cell markers (such as SOX2, Nanog, Oct4 and Stat3 ), so perhaps I will see some of these on display as well as its anti-IFT88 and anti-FXYD2 antibodies, which have both featured in two separate papers senior-authored by Fuchs and published in Cell [7 and 8].

Tuesday

On Tuesday I’ve picked a session I consider an alternative choice – “Clinical Trials with Functional Foods” (as food-based studies rarely feature on the blog!) Most of us have heard the saying “An apple a day keeps the doctor away”, but change ‘apple’ to ‘a serving of broccoli sprouts’ and ‘doctor’ to ‘cancer’ and you’ve got the basic gist of Thomas Kensler’s talk: “Interventions with broccoli sprout preparations in China”.

Kensler has done a lot of work into the biochemical and molecular mechanisms involved in both the induction of and protection against cancer in humans.  One of the biochemical pathways that interest his lab is the Keap1/Nrf2 signaling pathway, which is activated by several classes of chemopreventative agents – compounds proposed to inhibit, delay or reverse carcinogenesis.

Five day old organic broccoli sprouts. Source: Wikimedia Commons, Author: Julie Gibbons.

The symposium as a whole looks at cancer prevention trials that employ the nutrient components of whole foods and food-based products, as these may be an effective way of relieving the global burden of cancer in a cost-efficient manner. Other functional foods in the spotlight are anthocyanin-berry preparations found in blueberries, cranberries and bilberries to name a few, (so there you go, you can’t say you don’t get dietary advice from the Proteintech blog!)

Kensler’s lab used both Proteintech anti-Keap1 and anti-ALDH3A1 antibodies in a paper looking at the effects of Keap1 disruption and sulforaphrane treatment in human breast epithelial cells [9]. Sulforaphrane is an isothiocyanate found in cruciferous vegetables (cabbages, broccoli, cauliflower, etc.) and may be an effective, safe chemopreventive agent for use in humans.  The compound acts in part on the Keap1/Nrf2 pathway, regulating a battery of cytoprotective genes.

Wednesday

To round off my 2013 AACR Annual Meeting I’ve made a choice of three separate talks in the following sessions: “Emerging Targets in the Treatment of Lung Cancer”, “New Therapeutic targets for Ewing’s Sarcoma” and “Cancer Targets and Discovery”. Again these choices were for the most part inspired by the use of Proteintech antibodies by the sessions’ speakers, but they each have a similar theme: new therapeutic targets for cancer treatment.

Catch Matthew Meyerson’s “Genome alterations in non-small cell lung cancer”, Arul Chinnaiyan’s “Inhibition of ETS gene fusions in cancer” and William Hahn’s “Functional genomics to identify and validate cancer targets”, presenters who have published works using Proteintech antibodies in Nature (anti-MBIP) [10], Cell (PSMD2 and PSMB5 [11]) and The Journal of Cancer Discovery (KRAS) [12] respectively.

Final word

I hope that you can use this “guide” to supplement your own itinerary planning; however, you’re guaranteed to see some great science at the AACR Annual Meeting whatever you attend. Perhaps Proteintech antibodies have even played a role in some of the research on display? If you find any examples in addition to the ones presented here do let me know via Twitter!

See you in The District!

(@DebGrainger)

References

[1] Natrajan R. et al., Breast Cancer Res. 2012;14(2):R53

[2] Graeser M. et al., Clin Cancer Res. 2010;16(24):6159-68

[3] Hwang H.W. et al., Proc Natl Acad Sci U S A. 2009;106(17):7016-21

[4] Varfolomeev E. et al., J Biol Chem. 2008;283(36):24295-9

[5] Carpten J.D. et al. Nature 2007;448:439–444.

[6] Salhia B. et al., Hum Mutat. 2012 Aug;33(8):1216-27

[7] Ezratty E.J. et al., Cell 2011;145(7):1129-1141

[8] Lu C.P. et al., Cell 2012;150(1):136-150

[9] Agyeman A.S. Breast Cancer Res Treat. 2012; 132(1): 175–187

[10] Weir B.A. et al., Nature 2007;450(7171):893-8

[11] Nijhawan D et al., Cell. 2012;150(4):842-54

[12] Wang X.S. et al., Cancer Discov. 2011 Jun;1(1):35-43

*ER = estrogen receptor, PR = progesterone receptor

The post What to See at The AACR Annual Meeting 2013 | Proteintech’s Top Picks appeared first on The Proteintech Blog.

Prosaposin or PSAP, a glycoprotein encoded by the PSAP gene, can be cleaved into four products: saposins A, B, C, and D. All four are important for the hydrolysis of sphingolipids ― compounds known to play an important role in signal transmission and cell recognition.  Mutations in the PSAP gene can lead to a host of diseases including Gaucher disease (a lysosomal storage disease) and Tay-Sachs disease, a devastating condition that usually results in death by early childhood [1].

Prosaposin is crucially affected during and following cerebral ischemia, where insufficient blood flow in the brain disrupts its processing and thus glycosphingolipid metabolism.  This malfunction adversely affects synapses and cell signaling, and consequently normal brain function [2].  Correspondingly, the precursor molecule has been shown to possess neuroprotective effects and can act as an important growth factor, either being secreted or presented as an integral membrane protein. A study in the Journal of Neurochemistry found that prosaposin, when secreted by stromal cell-derived neuroprogenitor cells in the bone marrow, can protect adult neural cells from apoptotic cell death [3].

More recently the role of prosaposin in cell signaling has been highlighted in cancer research.  The anti-apoptotic protection offered by prosaposin has associated this pleiotropic growth factor with the promotion of proliferation of certain cancers. A recent study in the journal Cancer Science, has implicated PSAP expression in the promotion of breast cancer.  The authors of this paper used Proteintech’s anti-PSAP antibody in an enzyme linked immunosorbent assay (ELISA) to detect prosaposin secreted into the cell culture media. The assay detected decreased levels of prosaposin in cells transfected with plasmid designed to knock down PSAP expression [4]. The researchers in the Cancer Science study were able to show that prosaposin affects estrogen alpha-signaling in both in vivo and in vitro models of breast cancer. Decreased PSAP expression consequently led to a decrease in estrogen receptor alpha (ERa) expression and, accordingly, overexpression of this gene led to increased ERα levels.

Immunohistochemical staining of paraffin-embedded testis tissue using Proteintech’s PSAP antibody 10801-1-AP (10x lens).

The estrogen receptor plays an important role in estrogen hormone signaling ― a pathway that, when defective, contributes to the progression of certain kinds of estrogen-dependent breast cancers.  The findings of the Cancer Science study support the theory that activation of this receptor can be achieved independently of estrogen, with the help of growth factors like prosaposin [4]. In light of other findings that increased levels of prosaposin are associated with more aggressive forms of prostate cancer [5], such results suggest that prosaposin may be an important molecule in the protection of cancer cells, enabling them to survive and proliferate. Such work also presents prosaposin as a potential drug target in the development of stratified treatments for personalized cancer therapies.

Guest Blogger Profile

Kevin Measor is a PhD candidate in Neuroscience at the University of California, Riverside and an aspiring science writer. He studies how the  auditory cortex of the bat processes different properties of sound.  He also holds bachelors degrees in biology and history, and masters degrees in biology and education.  In his spare time he enjoys hanging out with his infant son, reading and birding.

Related Antibodies

PSAP rabbit polyclonal antibody – amino acid region 1-320 (Cat no. 10801-1-AP)

PSAP rabbit polyclonal antibody – amino acid region 60-142 (Cat no. 18396-1-AP)

PSAP rabbit polyclonal antibody – amino acid region 195-273 (Cat no. 18397-1-AP)*

PSAP rabbit polyclonal antibody – amino acid region 311-391 (Cat no. 18398-1-AP)*

PSAP rabbit polyclonal antibody – amino acid region 405-486 (Cat no 18423-1-AP)*

PSAP-like rabbit polyclonal antibody (Cat no. 55128-1-AP)

*Antibodies included in Proteintech’s current Buy One Get One Free promotion (ends 05/31/2013)

References

[1] S. Morimoto et al., Proc. Natl. Acad. Sci. 1990; 87(9):3493-3497

[2] W. J. Costain et al., Proteomics 2010; 10:3272-3291

[3] N. Li et al., J Neurochem 2010; 112:1527-1538

[4] Y. Wu et al., Cancer Sci 2012; 103(10):1820-1825

[5] S. Koochekpour et al., The Prostate 2012; 72:253-269

The post PSAP Antibody Uncovers Potential Role of Prosaposin in Cancer Survival appeared first on The Proteintech Blog.

Guest Post by Caroline Wood

Icam-1 (CD54) is a long established member of the immunoglobulin superfamily, expressed as a glycosylated transmembrane protein in leukocytes and endothelial cells. A key molecule in cell adhesion, it interacts with integrins such as LFA-1 and Mac-1 to stabilize cell-cell interactions. For example, it facilitates the passage of leukocytes between gaps in the endothelia (endothelial transmigration).

Recent work has uncovered Icam-1′s role in recruiting polymorphonuclear cells and macrophages to tumor sites; a process considered, initially, to be a part of the body’s defense mechanism against the invading tumor. However, studies in both pancreatic and hepatocellular carcinoma (HCC) have shown the opposite mechanism in action. Icam-1 appears to recruit immune cells that actively promote solid tumor survival and metastasis via angiogenesis [1]. This positions Icam-1 as an interesting new target for vacularization biomarkers and antivascular drugs [2].

Current antivascular therapies are not effective in preventing tumor growth entirely; rather, tumor growth is slowed. Therefore a complete understanding of tumor vascularization and the molecular components involved is needed to improve current anti-vascularization therapies.

Intermediate magnification micrograph of hepatocellular carcinoma, the most common form of primary liver cancer. Source: Wikimedia Commons, Author: Nephron.

Two recent papers, featuring Proteintech’s Icam-1 antibody, have contributed knowledge to our understanding of tumor growth and vascularization. The first, published in the journal of Gastroenterology and Hepatology in 2012, explores the role of cells originating from bone marrow in promoting angiogenesis in HCC. HCC takes the form of a solid tumor and vascularization is essential to its growth, metastasis and recurrence. The amount of HCC vascularization correlates tightly with disease progression and outcome. The authors of this work undertook ELISA and Western blotting experiments to track cell adhesion molecules and compare their expression in both pathological and healthy liver tissue samples. The profile of adhesion molecules in the pathological tissue showed recruitment of cells of a bone marrow-origin very early in tumor development, and this cell type increased in number with tumor growth [3].  This supports prior observations that the number of circulating bone marrow-derived cells is often raised during tumor growth and proliferation [4].

A further study identified  the cell type recruited to HCC tissue in the vascularization process as immature endothelial precursor cells (EPCs), these were found to gather and concentrate in HCC tissue, as determined via immunohistochemical staining. Immunohistochemical methods utilizing Proteintech’s Icam-1 antibody also demonstrated increased expression of Icam-1 in EPC-rich HCC tissue. The presence of Icam-1 is thought to contribute to further recruitment of additional EPCs [5].

Focusing on EPC-facilitated angiogenesis is a promising avenue in cancer research, potentially leading to tumor growth prevention. Such studies warrant further investigation into the roles bone marrow cells and cell adhesion molecules — such as Icam-1 — play in vascularization, and whether they present viable targets for the detection and treatment of liver cancer and other solid tumors.

More Icam-1 research

Icam-1 research has also focused on the role of this molecule in the inflammatory response; in particular, its role in brain injury — such as hemorrhage and stroke — has garnered interest. A 2004 study by Hu et al. showed that suppression of Icam-1 by sodium beta-aescin inhibited migration of neutrophils to sites of brain injury, following damage to the middle cerebral artery [6].  Consequently, this reduced inflammation and further damage to the surrounding brain tissue, highlighting Icam-1 as a potential drug target to lessen the effects of stroke and brain injury.

Further evidence for the role of Icam-1 in inflammation comes from the unlikely source of drug-induced gingival overgrowth. Gingivitus, also known as inflammatory gum disease, can be brought on by treatment with certain drugs, for example: during a course of treatment with the immunosuppressant Cyclosporin A. In these conditions, an increase in the expression of Icam-1 and production of interleukins in an inflammatory response have been observed. Phenytoin treatment is also associated with gingival overgrowth, but appears to downregulate production of Icam-1, suggesting cytokine disruption downstream of Icam-1 as the cause of overgrowth [7].

Guest Blogger Profile

Caroline Wood is a science writer based in Manchester, UK, with a background in genetics and neuroscience.

Related Proteintech antibodies

The featured Icam-1 antibody is Proteintech’s polyclonal rabbit Icam-1 (catalog number: 10020-1-AP). It was used in all of the cited studies – see below (excluding reference 4).

Other Proteintech Icam-1 antibodies:

Icam-1 rabbit polyclonal antibody (C terminal amino acid residues 352-575),  catalog no. 10831-1-AP

Icam-1 rabbit polyclonal antibody (C terminal amino acid residues 352-575),  catalog no. 15364-1-AP

Icam1 rabbit polyclonal (peptide antigen), catalog no. 16174-1-AP

Icam1 mouse monoclonal antibody (peptide antigen) catalog no. 60148-1-Ig

References

[1] Roland et al, Exp Biol Med (Maywood) 2010 Feb;235(2):263-70
[2] Arakawa et al, J Surg Res 2012 Jan;172(1):95-101.
[3] Zhu et al, J Gastroenterol Hepatol 2012 Jul;27(7):1241-51
[4] Goon et al., Neoplasia. 2006 February; 8(2): 79–88
[5] Sun et al, World J Gastroenterol. 2012 September 21; 18(35): 4925–4933.
[6] Hu et al, Acta Pharmacol Sin. 2004 Jul;25(7):869-75
[7] Suzuki et al, J Dent Res 2009 Dec;88(12):1131-6.

The post Tracking Cancer Vacularization and Progression with Proteintech’s Icam-1 Antibody appeared first on The Proteintech Blog.

Bcl2-associated athanogene (BAG3) (also known as CAIR-1) is a pleiotropic co-chaperone protein that is capable of activating multiple intracellular pathways. First identified as an anti-apoptotic protein, BAG3 has since been implicated in protein degradation via chaperone-mediated autophagy, CNS development, muscular dystrophies and neuropathies, the progress of viral infections, adhesion and migration, and cancer cell survival.

The BAG3 protein may also be of significance to research into neurodegenertion. Disruption to protein degradation pathways is known to contribute to diseases such as Parkinson’s, Alzheimer’s and Huntington’s. Interestingly, BAG3 is expressed in astrocytes in individuals with these conditions and facilitates clearance of protein aggregation during astrocytosis ― a response to CNS destruction [1]. Although a firm link to these diseases has not been established, BAG3 may represent a possible therapeutic target to inhibit disease progression during the early stages of neurodegenerative disease [1].

Work with Proteintech’s BAG3 antibody

There is evidence that mutated BAG3 causes a severe type of childhood muscular dystrophy (myofibrillar myopathy). Recent findings published in Nature Genetics support this body of work; the paper identifies this previously unrelated type of muscular dystrophy as a BAG3-related myopathy. Proteintech’s anti-BAG3 antibody was used in this study to examine whether the protein could interact with a mutated co-chaperone called DNAJB6. The antibody enabled the authors to describe the disease-aggravating effect of BAG3 interaction with pathological DNAJB6 in Limb-girdle muscular dystrophy (LGMD1D), suggesting a direct role for BAG3 in the pathomechanism of this disease [2].

Immunohistochemical detection of JC virus protein (brown staining) in a brain biopsy (glial cells infected with PML). Source: Wikimedia Commons, Author: Marvin 101. Disclaimer: detection antibody unknown.

Proteintech’s BAG3 antibody has also been utilized in work examining the function of BAG3 in response to viral infection. A recent paper examines the role of BAG3 in response to glial cell infection with JC virus (JCV), an infective agent known to possess oncogenic activity and also cause the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). With the Proteintech BAG3 antibody, the authors were able to show that BAG3 decreases viral expression of the large T‑antigen (T-Ag) protein, which gives the virus its malignant potential. BAG3 elicits its anti-T-Ag effects through induction of the oncogenic protein’s autophagic degradation [3]. In light of BAG3 playing a role in response to HIV-1 infection [4 and 5], such investigations demonstrate how BAG3 might play an important role in cellular response to viral infection through its involvement in apoptotic and autophagic processes.

Further applications of Proteintech’s BAG3 antibody

Proteintech’s BAG3 antibody (Catalog number 10599-1-AP) has also been employed to investigate both physiological and pathological BAG3 gene expression, the role of BAG3 in antigen processing, and aggresome targeted degradation via the dynein motor complex. The BAG3 antibody can be used for a number of applications including Western blotting, immunoprecipitation, immunohistochemistry and immunofluorescence.

BAG3 protein levels impact on apoptotic events in microglial cells harbouring HIV-1 virus. Previously, the authors had discovered BAG3 expression increased upon infection with HIV-1 [4], and subsequently when its expression was knocked down during these conditions in microglial cells, a marked increase in apoptosis was seen. After further investigation, the authors concluded that BAG3 protein induction counteracts cell stress and apoptotic events induced by HIV-1 infection [5].

Guest Blogger Profile

Katherine Campion is in the final year of her Physiology PhD at the University of Manchester, investigating the cell biology of calcium homeostasis and associated pathologies. Katherine has also spent time at Böhringer-Ingelheim in Vienna researching cancer signaling pathways. She is a published author in peer-reviewed journals and her work has received award at the 2010 Rank Prize Fund mini-symposia.

 

References

[1] K Seidel et al., Neuropathol Appl Neurobiol 2012;38(1):39-53

[2] J Sarparanta et al., Nat Genet 2012;44(4):450-5

[3] I K Sayrier et al. PLoS One 2012;7(9):e45000

[4] A Rosati et al., J Cell Physiol 2007;210(3):676-83

[5] A Rosati et al., J Cell Physiol 2009;218(2):264-7

The post BAG3 Antibody | Research Applications: from Muscular Dystrophies to Viral Infections appeared first on The Proteintech Blog.

CiteAb lists over 800,000 antibodies from 50 companies, along with the publications that cite them. CiteAb is the world’s largest independent citation-ranked antibody search engine, and is regularly updated with new antibodies and citations.

The search engine has been developed by a team based in Bath in the UK, and has been both supported and rigorously tested by the University of Bath. Following a period of testing and development in Beta CiteAb was launched this week as the world’s most comprehensive antibody search engine.

Dr Andrew Chalmers the founder of CiteAb said: “Citations are the best  guide to whether an antibody is likely to work in your laboratory, they are independent and easily verifiable. We’re aiming to make  CiteAb as comprehensive as possible and are delighted to be able to include Proteintech antibodies.”

Proteintech provides CiteAb with data for its primary antibodies. CiteAb is asking users of Proteintech antibodies to submit their publications to the site – increasing the profile of your work and helping others  to find the right antibody.

For more information visit www.citeab.com

The post New Antibody Search Engine: CiteAb appeared first on The Proteintech Blog.

Kelch-like ECH-associated protein 1 (Keap1) is a crucial substrate adapter protein for the Cul3-RBX E3 ubiquitin ligase complex, and a key regulator of the Nrf2 cytoprotective pathway. Under normal conditions, levels of Nrf2 are constantly regulated by rapid ubiquitination and proteasomal degradation; thus the antioxidant response is continually suppressed. However, when the cell is placed under oxidative stress, key cysteine residues in Keap1 are modified inhibiting the activity of the E3 ubiquitin ligase complex. Levels of Nrf2 are allowed to stabilize and increase, leading to the induction of numerous cytoprotective genes.

Considering its key role in cytoprotection, it is no surprise the Nrf2 pathway is of considerable interest to the field of cancer biology. As a consequence, Proteintech’s anti-Keap1 antibody has played a crucial role in a number of studies relating to cancer and chemotherapy.  In particular, dysregulation of the Nrf2/Keap1 pathway has been implicated in non-small cell lung cancer (NSCLC) [1].  Sequencing of KEAP1 revealed that 50% of NSCLC cell lines carry somatic mutations in this gene, with the mutations focused in the Kelch-like region of the protein ― the region responsible for Keap1’s E3 ligase repressor activity. In these cell lines, Keap1 is unable to inhibit the ubiquitination of Nrf2, and as a result they are unable to respond successfully to oxidative stress. In 2010, a study published in Clinical Cancer Research used Proteintech’s anti-Keap1 antibody to demonstrate that dysregulated Keap1 was particularly associated with poor outcome prognosis in NSCLC [2].

Immunohistochemical staining of paraffin-embedded human lung cancer using KEAP1 antibody 10503-2-AP (10x lens).

More recently, Proteintech’s anti-Keap1 antibody featured in a study published in the open-access journal PLoS One. This work successfully demonstrated that two known subtypes of NSCLC ― adenocarcinoma and squamous cell carcinoma ― can be considered as separate diseases with distinct pathogenic phenotypes [3]. This discovery naturally has considerable implications for the diagnosis and treatment of NSCLC, and their results support the idea of Keap1 as a molecular biomarker for subtypes of this disease.

The PLoS One study utilized Proteintech’s anti-Keap1 antibody to demonstrate that subtype-specific differences identifiable in the genome are also visible at the protein level through immunohistochemistry. Immunohistochemical analyses were carried out over a panel of 200+ lung tumors, and the antibody was used to identify whether Keap1 was inactivated in these cells. The results showed that dysregulation of Keap1 is a feature of adenocarcinoma, but that levels of Keap1 remain normal in squamous cell carcinoma. These results clearly identify Keap1 as a potential biomarker for identification of adenocarcinoma as a NSCLC subtype, and may have significant implications in future diagnosis and treatment of this disease.

More on Proteintech’s anti-Keap1 antibody

The anti-Keap1 antibody has also featured in a number of studies relating to cancer biology, including multiple myeloma, breast cancer and Wilms tumor, and other fields including inflammatory conditions. More details of these studies can be found in the following references: 21597922, 21821009, 22215675, 22222206, 22503981, 22629454, 23041549, 23285138, 21498569,21482715, 21465251, 21126175, 20427290, 20173742, 19279006

Guest Blogger profile

Katie Griffiths is a freelance scientific editor, technical writer and science communicator. She has recently graduated with a Research Masters in Structural Biology from The Institute of Structural and Molecular Biology at Birkbeck College. In her spare time she writes a blog The Molecular Circus, enthusing her readers about the molecular biology of everyday life.

Related Antibodies

Nrf2 rabbit polyclonal antibody (Cat no. 16396-1-AP)

Keap1 rabbit polyclonal antibody (Cat no. 10503-2-AP)

Keap1 mouse monoclonal antibody (Cat no. 60027-1-Ig)

References

[1] A. Singh et al., PLoS Med 3(10):e420 

[2] LM Solis et al., Clin Cancer Res 16(14):3743-53 

[3] WW Lockwood, PLoS One. 2012; 7(5): e37775 

The post NSCLC Research: Anti-Keap1 Antibody Helps Uncover Target’s Biomarker Potential appeared first on The Proteintech Blog.