International Journal of Scientific & Engineering Research, Volume 5, Issue 4, April-2014 1049

ISSN 2229-5518

Affinity purification of pbmc Tlr-4 of

Intestinal cancer patients

Ali Hasanain Ali

AbstractToll-like receptors (TLRs) play a critical role in host defense from microbial infection. TLRs recognize conserved molecular structures produced by microorganisms and induce activation of innate and adaptive immune responses. The inflammatory responses induced by TLRs play an important role TLRs not only in host defense from infection, but also in tissue repair and regeneration. This latter function of TLRs can also contribute to tumorigenesis. Recent findings show that functional TLRs are expressed not only on immune cells but also on cancer cells. TLRs play an active role in carcinogenesis and tumor progression during chronic inflammation that involves the tumor microenvironment. Damage-associated molecular patterns (DAMPs) derived from injured normal epithelial cells and necrotic cancer cells appear to be present at significant levels in the tumor microenvironment, and their stimulation of specific TLRs can foster chronic inflammation. These TLRs those are expresses on tumor cells are related to interactions between cancer cells, immune cells, and DAMPs through TLR activation in the tumor microenvironment.

This review discusses how the TLR is responsible for both providing immune response for various disease associated pathogens and how it involves in the carcinogenesis, cancer progression and metastasis

Index Terms— - Immune cells, cytokines , DAMPs, pathway , Tumor Angiogenesis , PAMP.

—————————— ——————————


TLRs are evolutionary conserved from plants to verte- brates. In mammals there are 12 identified TLRs. These recep- tors undergo homo or hetero dimerization to detect a wide range of PAMPs (Pathogen Associated Molecular Pattern) in- cluding lipids, lipoproteins, proteins, glycans, and nucleic ac- ids. These receptors are the prime pathogen sensing gates in the body.
TLRs play essential roles in the innate immune re-
sponses to microbial pathogens based on their ability to rec- ognize pathogen-associated molecular patterns (PAMPs) (Aki- ra et al., 2006).
Till date in humans 10 different TLRs have been identified.TLRs1-9 are conserved between humans and mice.
In addition, TLR10 is expressed in humans but not in mice, whereas TLR11 is present in mice but not in humans.
TLRs1, 2, 4, 5 and 6 are primarily located on the cell surface and recognize bacterial components. TLRs3, 7, 8 and 9 are generally located in the endocytic compartments and pri- marily recognize viral products.
This study shows that the immune system, particular- ly the innate immune system, has a skillful means of detecting invasion by microorganisms. Subsequently, mammalian hom- ologues of Toll receptor were identified one after another, and designated as Toll-Like Receptors (TLRs). Functional analysis of mammalian TLRs has revealed that they recognize specific patterns of microbial components that are conserved among pathogens, but are not found in mammals.
•Ali hasanain Ali. is currently pursuing masters degree pro- gram in physiology,department of basic medical science, Nursing College,University of Almuthanna, Iraq.
In signaling pathways via TLRs, a common adaptor, MyD88, was first characterized as an essential component for the acti- vation of innate immunity by all the TLRs. However, accumu- lating evidence indicates that individual TLRs exhibit specific responses. Furthermore, they have their own signaling mole- cules to manifest these specific responses.

Toll-Like Receptors (TLRs) have been established to play an essential role in the activation of innate immunity by recognizing specific patterns of microbial components (Ta- ble.1). shows TLR signaling pathways arise from intra- cytoplasmic TIR domains, which are conserved among all TLRs.
Recent accumulating evidence has demonstrated that
TIR domain-containing adaptors, such as MyD88, TIRAP, and
TRIF, modulate TLR signaling pathways. MyD88 is essential
for the induction of inflammatory cytokines triggered by all
TIRAP is specifically involved in the MyD88-
dependent pathway via TLR2 and TLR4, whereas TRIF is im-
plicated in the TLR3- and TLR4-mediated MyD88-
independent pathway. Thus, TIR domain-containing adaptors provide specificity of TLR signaling.
Infection, inflammation, and injury may converge to increase the risk of cancer in a multitude of ways. Microbial colonization can in some cases promote tumorigenesis. For
example, Helicobacter pylori (Schneider et al.,2009) and hepa- titis C virus (HCV) ( Ning XU et al.,2008) may occupy host niches that lead to chronic inflammation due either to infec- tion or sterile injury is an important risk factor for cancer. The inflammatory response is well known to play a critical role in all stages of cancer development including initiation, promo- tion, and progression. In addition, regardless of the origin,

IJSER © 2014

International Journal of Scientific & Engineering Research, Volume 5, Issue 4, April-2014 1050

ISSN 2229-5518

whether it be due to infection, inflammation, irritation, or on- cogene or DNA-damage associated apoptosis, there is a great deal of cell death and tissue injury associated with cancer. In- deed there are interesting parallels between tumorigenesis, tissue repair, and regeneration. Parallel to the recognition of the importance of TLRs as sensors and shapers of the overall anti-tumor response, TLRs have emerged as an important ap- plication area and focus of basic research and applied to de- velopment of cancer therapeutic and vaccine research.
The tumor microenvironment, which includes cancer
cells, stressed normal cells, stromal tissue and extracellular
matrix, has recently been implicated as a major factor for pro-
gression and metastasis of cancer.
Recent studies show that activated TLRs expressed on can-
cer cells can dampen the anti-tumor functions of infiltrating immune cells, thereby altering the inflammatory response in a manner that promotes cancer progression.

Table.1. TLR Recognition of Microbial Components

The activation of TLR signaling pathways originates from the cytoplasmic TIR domains.
In the signaling pathway downstream of the TIR domain, a TIR domain-containing adaptor, MyD88, is present which plays a crucial role in signal transduction.
Recent studies shows that there are two types of
pathway for this TLR mediated signaling. First one is Myd88
dependent Pathway and second one is Myd88 independent
pathway (Takeda et al., 2003).

1.2. TLRs and Mechanisms of Tumorigenesis

TLR recognize and respond to exogenous and en- dogenous ligands through signaling pathways leading to in- flammatory cascade mediator production which direct the innate and adaptive immune response. It is increasingly rec- ognized that inflammatory processes play a key role in tumor- igenesis. TLRs, as in other human diseases, appear to act as double edged swords in tumorigenesis. Overall, research stud- ies suggest that TLRs as a family are involved in both inhibit- ing and promoting cancer.

1.3. How TLR expressed in cancer cells Signals to Carcino- genesis

There is accumulating and steadily growing evidence that cells of several human malignancies express single or more commonly multiple TLRs.There are, however several lines of evidence suggesting biological impact of TLRs expres- sion on tumor cell growth and survival. Different TLRs ex- pressing on cancer cells involve in different tumor progression (Table.2). There is also evidence that bacteria present in the tumor microenvironment are able to promote tumor growth via TLR signaling.

Table.2. Type of Cancer and Involvement of TLR. (Swantek et al.,2000)

(Cell 124, 783–801, February 24, 2006 ª2006 Elsevier Inc.)

1.1. General Mechanism of Action Of TLRs In creating Im- mune Response

Cancer-associated fibroblasts (CAFs) are important compo- nents of the tumor microenvironment, and they are the main

IJSER © 2014

International Journal of Scientific & Engineering Research, Volume 5, Issue 4, April-2014 1051

ISSN 2229-5518

cellular component of the tumor stroma. Unlike normal fibro- blasts, CAFs are perpetually activated. (Shimoda et al.,2010).Their origin is not well understood, but they appear to be as important as immune cells in the tumor microenvi- ronment. A recent study proposed that TGFβ has a crucial role in activation of CAFs. Activated CAFs promote the prolifera- tion and progression of cancer through the production of growth factors and metalloproteinases.Therefore, a TLR- related increase in TGFβ might lead to assembly and activa- tion of CAFs in the tumor microenvironment. In summary, during cancer progression in the setting of chronic inflamma- tion, TLR ligands activate, TLRs expressed in cancer cells. Ac- tivated cancer cells release cytokines and chemokines that are an important component of the tumor microenvironment. Cy- tokine-activated infiltrating immune cells subsequently can induce further cytokine release that contributes to activation of CAFs and impairs the function of APCs, effector T-cells and TAA-specific immunity; possibly resulting tumor immunotol- erance. The interplay and additive effects of these events facili- tate continuous activation of TLR in cancer cells or adjacent normal epithelial cells, thereby maintaining a hostile tumor microenvironment and promoting tumor progression.

1.4. Tumor Angiogenesis and TLR

TLRs also seem to have an important role in tumor angiogenesis, i.e., the formation of new capillary blood vessels from existing vessels outside of the tumor. The developing tumor depends on angiogenesis as a source of more oxygen and nutrients for survival and growth. Vascular endothelial growth factor (VEGF) is the main factor involved in tumor angiogenesis (Reinmuth et al.,2001) and is part of the aberrant molecular pattern associated with TLR signals. VEGF is se- creted by cancer cells directly and by immune cells and CAFs. New vessels induced by VEGF are abnormal: they are hetero- geneous in distribution, irregular in shape, and not organized into arterioles, venules and capillaries. Their varied permeabil- ity leads to high interstitial pressures and further hypoxia, which stimulates additional VEGF production. Hypoxia char- acterizes solid tumors; it is a stress factor that might cause cells to release DAMPs. These ligands activate TLR signals and con- tribute to the aberrant molecular pattern in the tumor micro- environment. (Fig.1).
The TLR contribution to tumor angiogenesis has been
investigated in H. pylori-associated gastric cancer (Chang et al.,
2005).This study reported that H.pylori induced COX-2 expres-
sion and PGE2 release enhanced tumor angiogenesis via
Y. Sato et al., Cancer Microenvironment (2009

Fig.1. Tumor microenvironment and Angiogenesis

1.5. Disruption of Anti-tumor Response of TLR Expressed in Immune cells

Under normal conditions, scheduled cell death is reg- ulated by adenosine triphosphate (ATP) and related apoptotic pathway factors; this regulation drives fragmentation of cellu- lar macromolecules and the speedy subsequent phagocytosis and clearance of apoptotic debris. However, in cancerous con- ditions, cells dying by non-apoptotic pathways, principally necrosis, release DAMPs into the extracellular space. DAMPs are nuclear or cytosolic proteins with defined intracellular functions but different extracellular actions after cytolysis.
DAMPs released from injured or dying cells are rec- ognized by TLRs on immune cells; subsequent TLR signals disrupt the anti-tumor immune response and lead to cancer progression (Carta et al,.2009).
Candidate DAMPs include heat shock proteins (HSP
60, 70), ATP and uric acid, the S100 family of calcium modu- lated proteins, nuclear protein high-mobility group box 1 (HMGB1), and nucleic acids. HMGB1, a DNA binding protein, is one of the best-characterized DAMP. HMGB1 regulates in- tracellular transcription and mediates extracellular proin- flammatory processes. HMGB1 released during unscheduled cell death activates an immune response via TLR signals. Dur- ing tumor expansion, nucleic acids released from necrotic can-

IJSER © 2014

International Journal of Scientific & Engineering Research, Volume 5, Issue 4, April-2014 1052

ISSN 2229-5518

cer cells or adjacent injured normal epithelial cells also act as DAMPs. The high rate of unscheduled cell death in the tumor microenvironment elevates nucleic acid DAMPs. Elevated levels of nucleic acid DAMPs and other DAMPs might foster chronic inflammation, a hallmark of the tumor microenviron- ment.
SDS PAGE of protein and comparison with protein molecular weight marker for the determination of the protein bands re- sponsible for the TLR expression in cancer patients

3. Material and Methods

Figure 2 shows how interactions between TLRs and
DAMPs could create and maintain a self-perpetuating tumor

microenvironment. In this microenvironment, cancer cell death might stimulate cancer progression if nucleic acid frag- ments released by the dead tumor cells are transfected into normal cells, thereby changing the normal cell’s properties. Normal cells in the tumor microenvironment might also be transfected by microRNA released from tumor cells, because these small RNA molecules (20–22 base pairs) are easily taken up by cells. Horizontal mediated transfection of microRNA and mRNA in mammalian cells is an intriguing possibility but has yet to be demonstrated in vivo. This phenomenon could explain the expression of tumor-related proteins by normal cells in the tumor microenvironment.

Fig.2. Interaction between TLR, DAMP and PAMP

2 The objectives of our work is_

1 . Collection of blood from healthy and cancerous patients.
2. Density gradient centrifugation for Separation of PBMC (Peripheral Blood Mononuclear Cells) from blood by using ficoll.
3. Extraction of protein from PBMC.
4. Affinity Chromatography
TLR Protein Isolation Buffer Isolation from Blood Sample
Preparation of HBSS Solution (Hank’s balanced salt solution)

3.1 Requirements:

• Blood
• HBSS solution
• Ficoll

3.2 Affinity Chromatography:

Two phases of Affinity Chromatography
• Stationary Phase
• Mobile Phase




• All the vertical gel apparatus was washed by spirit.
• The gel casting tray was prepared by using the 3 spacer and join the glass by silica gel.
• After casting the vertical gel apparatus, separating gel was loaded (up to 5cm long) between the glass plates and allowed to solidified.
• Stacking gel was poured on the top of the separating gel.
• The comb was inserted immediately into the stacking gel and allowed the gel to set.
• Protein sample with the sample buffer was taken in the ratio 1:1.
• The sample was boiled for 2minutes in the water bath.
• After settling of the stacking gel the comb was re- moved slowly.
• Water molecules were dried from the wells by what- mann filter paper.
• The bottom spacer was removed and the gel slab was placed in the buffer in the vertical gel apparatus.
• The sample was loaded into the wells.
• Powers of 50 volts was supplied to the apparatus and allows the sample to run in the gel.

IJSER © 2014

International Journal of Scientific & Engineering Research, Volume 5, Issue 4, April-2014 1053

ISSN 2229-5518

• The tracking dye bromophenol blue when reaches at the bottom of the gel the current was turned off.
• The gel was removed slowly from between the glass plates and put in coomassieve blue staining solution for overnight.
• The gel was washed in the destaining solution till a clear back ground comes.
• Only the stained proteins were visible as blue colour bands.


The complex formed between Toll receptor TLR4 and myeloid differentiation factor MD2 defines a major cell surface receptor for lipopolysaccharide (LPS), a gram- negative bacterial antigen that has been implicated in infec- tious complications. In our present study we have isolated the PBMC (peripheral blood mononuclear cells) from normal and Intestinal cancer blood samples. We extracted the total pro- teins from PBMC through ultrasonication by dissolving the cells in appropriate protein extraction buffer.SDS PAGE has done for the qualitative analysis of the protein. We compared the cancerous blood sample with the protein molecular weight marker.
KDa. We compared these results with the data of the molecu- lar weight of TLR of a normal person. So as per the data it
TLR-1 84KDa
TLR-2 84KDa
TLR-3 97KDa
TLR-4 90KDa

TLR-5 91KDa TLR-6 91KDa TLR-7 21KDa TLR-8 120KDa TLR-9 116KDa TLR-10 95KDa
Then, from the total protein, purification has done by affinity chromatography for TLR-4 by fixing E.coli cells in the stationary phase. As E.coli cells outer membrane consists of Lipopolysachcharides(LPS).As it can be used for TLR-4 to bind to the stationary phase. Collection of TLR-4 from the sta- tionary phase has done by using buffer Tris Nacl. In this buff- er the concentration of Tris is 10mM with a pH 8 and the con- centration of Nacl is 200mM.

Fig: 3 Comparison of Cancerous protein sample with Pro- tein Marker

According to the above data for human TLR family, we didn’t get any protein band above 97KDa.The bands for
90KDa may be of the presence of TLR 4 and Bands coming near 91KDa
the presence of TLR 5/6.

From the results we got as TLR 3, 4, 5 and 6 are
expressing at same level both in normal and intestinal pa-
tients.As it is expressing in both normal and cancerous blood,
for further studies we have to study whether the protein is
coming in its native structure by native gel electrophoresis in cancerous blood sample. Further analysis of TLR-4 in cancer can be done by Western blotting.
TLR-4 from intestinal can- cer patient’s blood

Fig: 2 TLR-4 Protein Purified Affinity chromatography

From the SDS PAGE of eluted sample it has found the bands are detected near to 90 KDa, in between 97and 66

IJSER © 2014

International Journal of Scientific & Engineering Research, Volume 5, Issue 4, April-2014 1054

ISSN 2229-5518

Fig:4 TLR Protein Isolated from Normal Blood


TLRs are expressed on many types of cancer cells, tumor stromal cells and infiltrating immune cells. TLR activa- tion during inflammation and injury plays an active role in the surrounding microenvironment. Similarly, in carcinogenesis and tumor progression TLRs play an active role in the tumor microenvironment. During chronic inflammation, abnormal activation of TLRs in normal fibroblasts and epithelial cells might facilitate neoplastic transformation and carcinogenesis. Cancer cells activated by TLR signals can release cytokines and chemokines that recruit and optimize immune cells to release further cytokines and chemokines. The result is an ab- errant cytokine profile associated with immune tolerance, can- cer progression and propagation of the tumor microenviron- ment. DAMPs derived from injured normal epithelial cells and necrotic cancer cells appear to be present at significant levels in the tumor microenvironment, and their stimulation of spe- cific TLRs might foster chronic inflammation.
This mechanism is complex and thus far not well under-
stood; however, it is clear that carcinogenesis, cancer progres-
sion, and site specific metastasis are related to interactions between cancer cells, immune cells, DAMPs and PAMPs through TLR signals in the tumor microenvironment. Better understanding of these signals and pathways will lead to de- velopment of novel therapeutic approaches to a wide variety of cancers.


[1] Akira S., Satoshi., Uematsu and Takeuchi O., 2006. Pathogen recogni- tion and innate immunity research institute for microbial diseases. Cell; Vol.124: Page -783-801.

[2] Bin LH., Xu LG., Shu HB.,2003. TIRP a novel TIR domain-containing adapter protein involved in Toll/interleukin-1 receptor signaling. Journal of Biological Chemistry; Vol.278: Page 24526–32.

[3] Brief report Blood plasmacytoid dendritic cell responses to CpG

oligodeoxynucleotides are impaired in human newborns

[4] Bruno L., Nicolas E., Michaut L., Reichhart J-M., Hoffmann JA.,1996.

The dorsoventral regulatory gene cassette spatzle/Toll/cactus con- trols the potent antifungal response in Drosophila adults.Cell; Vol.86: Page 973– 83.

[5] Carta S., Castellani P., Delfino L., Tassi S., Vene R and Rubartelli A.,2009.DAMPs and inflammatory processes: The role of redox in the different outcomes. Journal of Leukocyte Biology; Vol. 86: 549–555.

[6] Chang Ya-Jen., Ming-Shiang Wu., Lin J.T and Ching-Chow Chen.,2005. Helicobacter pylori-induced invasion and angiogenesis of gastric cells is mediated by cyclooxygenase-2 induction through TLR2/TLR9 and promoter regulation. Journal Of Immunology; Vol.175: Page 8242-8252.

[7] David S., Schneider., Kathy L., Hudson., Ting-Yi Lin and Kathryn V.,1991. Anderson dominant and recessive mutations define func- tional domains of Toll, a transmembrane protein required for dorsal- ventral polarity in the drosophila embryo. Genes & development; Vol.5: Page 797-807.

[8] Deng L., Wang C., Spencer E., Yang L., Braun A., You J et al.,2000.

Activation of the IÉ B kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell; Vol.103: Page 351–61.

[9] Dominique De Wit, Ve´ronique Olislagers, Stanislas Goriely, Franc¸oise Vermeulen, HermannWagner, Michel Goldman, and Fa- bienne Willems

[10] Ellis.,2001.Induction of VEGF in perivascular cells defines a poten- tial paracrine mechanism for endothelial cell survival. The FASEB Journal. Vol. 15.

[11] Hashimoto C., Hudson KL., Anderson KV.,1988. The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. Cell; Vol.52: Page 269–79.

[12] Kaori Fuse, Kyoko Katakura, Natsumi Sakamoto, Hiromasa Ohira Toll-like receptor 9 gene mutations and polymorphisms in Japanese ulcerative colitis patients

[13] Kawai T., Takeuchi O., Fujita T., Inoue J., Muhlradt PF., Sato S et al.,2001. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IRF-3 and the expression of a subset of LPS-inducible genes. Journal Of Immunology; Vol 167: Page 5887–94.

[14] Krishnan J., Kumar S., Tsuchiya M., Gwang L and Choi S., 2007. Toll- like receptor signal transduction. Experimental and molecular medi- cine, Vol. 39, No. 4, Page 421-438,

[15] Ning XU., Hang-ping YA., Zhen SUN., Zhi CHEN., 2008.Toll-like receptor 7 and 9 expression in peripheral blood mononuclear cells from patients with chronic hepatitis B and related Hepatocellular carcinoma.Acta Pharmacologica Sinica; Vol 29: Page 239-244.

[16] Protein Kinase D1: A New Component in TLR9 Signaling, Jeoung- Eun Park, Young-In Kim and Ae-Kyung Yi J. Immunol.


[17] Reinmuth N., Wenbiao Liu., Young D., Jung., Syed A., Ahmad., Raymond M., Shaheen., Fan Fan., Corazon D., Bucana., Gerald Mcmahon., Gary E., Gallick., And Lee M.

IJSER © 2014

International Journal of Scientific & Engineering Research, Volume 5, Issue 4, April-2014 1055

ISSN 2229-5518

[18] Sato Y., Goto Y., Norihiko Narita., Dave S.B Hoon., 2009. Cancer cells expressing toll-like receptors and the tumor microenvironment. Can- cer Microenvironment; Page S205–S214.

[19] Schneider N., Krishna U., Romero-Gallo J., Dawn A., Israel.,. Blanca Piazuelo M., Constanza Camargo M., Liviu A., Sicinschi., Barbara G., Schneider, Pelayo Correa., and Richard M., Peek Jr., 2009. Role of Helicobacter pylori CagA Molecular Variations in Induction of Host Phenotypes with Carcinogenic Potential, Journal of Infectious Dis- ease: Page 1218–1221.

[20] Swantek JL., Tsen MF., Cobb MH., Thomas JA.,2000. IL-1 receptor associated kinase modulates host responsiveness to endotoxin. Jour- nal Of Immunology; Vol 164: Page 4301–6.

[21] Toll-like receptors stimulate human neutrophil function Fumitaka

Hayashi, Terry K. Means, and Andrew D. Luster

[22] Wang C., Deng L., Hong M., Akkaraju GR., Inoue J-I., Chen ZJ., 2001.

TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature; Vol.412: Page 346–51.

IJSER © 2014