Loading...

Summer Research Fellowship Programme of India's Science Academies

Investigating the role of HEDGEHOG signaling pathway in regulation of CD133, CD44, OCT4 expression (colorectal cancer stem cell markers)

Faisal Jamal

Aligarh Muslim University, Aligarh

Guided by:

Prof M. Radhakrishna Pillai

Director, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695014

Abstract

Most cancers, including colorectal cancer, are believed to harbor a pool of cancer stem cells. Cancer stem cells play a major role in cancer development because of self-renewal, chemo resistance and tumorigenic capacity. They are also known to have a role in the initiation, progression, and recurrence of cancer. Various signaling pathways are believed to be involved in the onset and progression of colorectal cancer. Hedgehog pathway (Hh) is one of them. Hh pathway is essential for renewal of CSCs and is aberrantly activate in a variety of cancers. The classic inhibitor of Hh pathway is Cyclopamine. Cyclopamine inhibits Smo receptor in membrane. Here we have investigated the role of Hedgehog pathway in regulation of CD133, CD44, OCT4 (cancer stem cell markers) expression. The CD133 (also called Prominin-1) is believed to be associated with tumorigenicity and progression of the disease. The up-regulation of CD133 in colorectal cancer correlated strongly with poor prognosis and synchronous liver metastasis. Although the precise role of CD133 is unknown, the goal of the study was to clarify the possible role of Hh signaling pathway's genes in the regulation of CSCs and the effect of SANT1 on CSCs markers. Gene expression of the specific markers are analyzed by Real time q-PCR at mRNA level and Western Blotting at protein level from the cells with and without Hh inhibitor (SANT1). After treatment with SANT1, we observed a decrease in the expression of cancer stem cell markers as well as Hh effectors GLI1 and GLI2. Further studies are going on in order to find a better concentration of SANT1 and analyzing the role of Hh pathway in regulation of cancer stem cell markers.

Keywords: Colorectal Cancer, Cancer stem cells, Hedgehog pathway, SANT1,

Abbreviations

APCAdenomatous Polyposis Coli
CIMPCpG Island Methylator Phenotype
CINChromosomal Instability Pathway
CRCColorectal Cancer
CSCCancer Stem Cell
DMEMDulbecco's Modified Eagle's Medium
EDTAEthylenediaminetetraacetic acid
FBSFoetal Bovine Serum
FZDFrizzled
PBSPhosphate Buffered Saline
PVDFPolyvinylidene difluoride
SDS PAGESodium dodecyl sulphate polyacrylamide gel elctrophoresis
SHHSonic Hedgehog
SUFUSuppressor of fused

INTRODUCTION

Cancer

Cancer is a complex disease occurring as a result of a progressive accumulation of genetic aberrations and epigenetic changes that enables them to escape from normal cellular and environmental controls. Normal cells are constantly subjected to signals that develop a degree of autonomy from these signals, resulting in uncontrolled growth proliferation. An isolated abnormal cell that does not proliferate more than its neighbours does no significant damage; but if its proliferation is out of control, it can give rise to a tumor or neoplasm. Neoplastic cells can have numerous acquired genetic abnormalities including aneuploidy, chromosomal rearrangements, amplifications, deletion, gene rearrangements and loss of function or gain of function mutations. As long as the neoplastic cells remain clustered together in a single cell mass, the tumor is said to be benign. A tumor is considered cancer only if it is malignant.

Cancers are classified based on the tissue and cell type from which they arise. Cancers arising from epithelial cells are termed as carcinomas. This group includes many of the most common cancers and include nearly all those in the breast, prostate, lung, pancrease and colon. Sarcoma is termed as cancers arising from connective tissue. Lymphoma and Leukemia: These two classes arise from haemopoietic cells. Germ cell tumors are cancers derived from pluripotent cells, most often presenting in the testical or the ovary. Blastoma is cancer derived from immature precursor cells or embryonic tissue.

Colorectal cancer

Colorectal cancer (CRC) is a type of tumor called adenocarcinoma. It is the most commonly diagnosed cancer among both men and women, caused by inherited or acquired mutations most probably in the intestinal crypt stem cell. Colorectal cancer begins when healthy cells in the linning of the colon or rectum transform and grow out of control, forming a mass called tumor. Colorectal cancer most often begins as a polyp, noncancerous growth that may develop on the inner wall of the colon or rectum as people grow older. If not treated or removed, a polyp can become a life threatening cancer. Recognizing and removing pre-cancerous polyps can prevent colorectal cancer. There are several forms of polyps. Adenomatous polyps, or adenomas, are a form of growth that may become cancerous. Hyperplastic and inflammatory polyps, usually do not carry a risk of developing into cancer. However, large hyperplastic polyps, especially on the right side of the colon, are of concern and should be completely removed. Colorectal cancer may also develop from areas of abnormal cells in the lining of the colon or rectum. This area of abnormal cells is dysplasia and is more commonly seen in people with certain inflammatory diseases of the bowel such as Crohn's disease or ulcerative colitis.

The pathogenesis of colorectal cancer is very complex and diverse and is also influenced by multiple factors. A progression from normal mucosa to adenoma to carcinoma was supported by the demonstration of accumulating mutations in genes of K-ras, adenomatous polyposis coli (APC), Tumor protein P53 (TP53), and deletion in colorectal carcinoma (DCC). Major canonical pathways altered in colorectal cancer are chromosomal instability pathway, microsatellite instability pathway (CIN), characterized by widespread loss of heterozygosis (LOH) and gross chromosomal abnormalities.

The Wnt, Hedgehog and Notch pathways are inherent signaling pathways in normal embryogenesis, development, and hemostasis. These signaling pathways are implicated in both cancer and stem cells. Dysfunction of these pathways are evident in multiple tumor types and malignancies. Specifically, aberrant activation of these pathways is implicated in modulation of cancer stem cells (CSCs).

The Sonic Hedgehog (Hh) pathway is essential for normal embryonic development and plays a crucial role in adult tissue maintenance, renewal, and regeneration. In addition to vital roles during normal embryonic development and adult tissue homeostasis, aberrant Hh signaling is responsible for the initiation of a growing number of cancers including basal cell carcinoma, edulloblastoma, and rhabdomyosarcoma; more recently over reactive Hh signaling has been implicated in pancreatic, lung, prostate and breast cancer. Thus, understanding the mechanisms that control Hh signaling activity will inform the development of novel therapeutics to treat a growing number of Hh-driven pathologies.

The advent of small-molecule inhibitors for targeting these pathways and their success in other diseases, either as single agents or in combination therapy, provides a rationale for exploring these pathways as potential targets in the treatment of colon cancer.

The classic inhibitor of Hh pathway is Cyclopamine, a naturally occuring teratogenic alkaloid. It disrupts the cholesterol bio-synthesis and specifically antagonizes the Shh signaling pathway through direct interaction with Smoothened (SMO), which functions upstream of GLI1.

Objectives of the Research

Overall objective

  • Evaluating the effect of SANT1 (Hh pathway inhibitor) on HT-29 cell line.
  • Investigating the role of Hh signaling pathway in regulation of expression of CD133, CD44, OCT4 expression (cancer stem cell markers).

LITERATURE REVIEW

PATHOGENESIS OF CANCER

Cancer is driven by genetic and epigenetic alterations that allow cells to proliferate and escape mechanisms that normally control their survival and migration. Many of these alterations map to signaling pathways that control their survival and migration. And many of these alterations map to signaling pathways that control cell growth and division, cell death, cell fate, and cell motility. Mutations that convert cellular proto-oncogens to oncogens can cause hyper activation of these signaling pathways, whereas inactivation of tumor suppressors eliminates critical negative regulators of signaling. Proto-oncogens are physiological regulators of cell proliferation and differentiation while oncogens are characterized by the ability to promote cell growth in the absence of normal mitogenic signals. Tumor suppressor genes commonly contribute to the fidelity of the cell cycle replication process. They may act as negative regulators of oncogens, cell cycle check points. Mutations in tumor suppressor genes are loss-of -function mutations and so occur in both the alleles in a gene.

In solid tumors, these alterations typically promote progression from a relatively benign group of proliferating cells (hyperplasia) to a mass of cells with abnormal morphology, cytology, appearance and cellular organization. After a tumor expands, the tumor core loses access to oxygen and nutrients, often leading to the growth of new blood vessels (angiogenesis), which restores the access to nutrients and oxygen. Subsequently, tumor cells can develop the ability to invade the tissue beyond their normal boundaries, enter the circulation, and seed new tumors at other location (metastasis), the defining feature of malignancy (​Sever and Brugge 2015​). During the course of tumor progression, cancer cells can acquire a number of characteristic alterations. These include the capacity to proliferate independently of exogenous growth-promoting or growth-inhibitory signals, to invade surrounding tissues and metastasize to distant sites, to elicit an angiogenic response and to evade mechanisms that limit cell proliferation, such as apoptosis and replicative senescence. These properties reflect alterations in the cellular signaling pathways that control cell proliferation, motility, and survival. Many of the proteins currently under investigation as possible targets for cancer therapy are signaling proteins that are components of these pathways. (​Martin 2003​)

HALLMARKS OF CANCERS

The hallmarks of cancer comprise of six biological capabilities acquired during the multistep development of human tumors. They include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. Underlying these hallmarks are genome instability, which generates the genetic diversity that expedites their acquisition, and inflammation, which fosters multiple hallmark functions. Conceptual progress in the last decade has added two more emerging hallmarks of potential generality to this list – reprogramming of energy metabolism and evading immune destruction. In addition to cancer cells, tumors exhibit another dimension of complexity; they contain a repertoire of recruited, apparently normal cells that contribute to the acquisition of hallmark traits by creating the "Tumor microenvironment" (​Hannahan & Weinberg 2011​).

Hallmarks4.jpg
    Hallmarks of cancer

    COLORECTAL CANCER

    Colorectal cancer (CRC) is the second most commonly newly diagnosed cancer and accounts for the third highest number of cancer related deaths worldwide and of increasing importance in Asia (​Al-Sohaily et al 2012a​). Colorectal cancer is one of the major causes of morbidity and mortality, representing the second major cause of cancer incidence among females and the third among males.

    PATHOGENESIS OF COLORECTAL CANCER

    The pathogenesis of CRC is very complex, diverse and is also influenced by multiple factors, some of which are related to diet and lifestyle, while others are related to genetic predisposition (​Janindra Warusavitarne, 2012​). Another risk factor is the presence of long-standing inflammatory bowel diseases (IBD), either Crohn's or ulcerative colitis (​Colussi et al 2013a​).

    Research conducted during the past 30 years has demonstrated the existence of at least three pathways; chromosomal instability, microsatellite instability (CIN) pathway, characterized by widespread loss of heterozygosis (LOH) and gross chromosomal abnormalities. (​Chang-Woo Lee, 2003​) The second involves approximately 15% of CRC and is due to derangement of the DNA Mismatch Repair (MMR) system and consequential microsatellite instability (MSI). The MMR system is responsible for the production of proteins that recognize and direct repair of single nucleotide mismatches at microsatellite sequences that escape the proofreading system of DNA polymerase (​Colussi et al 2013a​). Concordant methylation of the CG di-nucleotide in the promoter region of multiple genes is called CpG Island Methylator Phenotype (CIMP). Inflammations can also cause carcinogenesis. Classification of CRC based on the presence of MSI and CIMP describes five molecular subtypes, each with a different molecular profile and clinico-pathological features. These are given below in the Table 1.

     MOLECULAR SUBTYPES OF CRC 
    MOLECULAR SUBTYPES OF CRCMUTATED GENE%
    CIMP high/MSI highBRAF mutation and MLH1 methylation12% of CRC
    CIMP high/MSI low or microsatelliteBRAF mutation and methylation of multiple genes.8%
    CIMP low/MSI low or microsatellite stableChromosomal instability (CIN), K-ras mutation and MGMT methylation20%
    CIMP negative/microsatellite stableOriginates in traditional adenoma and is characterized by CIN57%
    hereditary Non polyposis Colorectal cancerCIMP negative/MSI high; negative for BRAF mutations

    Importantly, new studies have shown that micro RNAs can also contribute to colorectal carcinogenesis (​Luigi Ricciardiello, 2013​). Various miRs, including miR-101, the let7 family, miR-126, and miR-142-3P, have been found to act as tumor suppressors. Some miRs are highly expressed in CRC cells, playing a major role in creating a microenvironment that allows the cancer cells to thrive. These miRs are known as oncomirs. One of the earliest miRs to be identified as an oncomir was miR-21, which plays an important role in the initiation, progression, and metastasis of CRC (​Mohammadi et al 2016​). The adenoma to carcinoma transition is determined firstly by the K-ras gene, a proto-oncogene that encodes for the GTPase protein involved in the transduction and propagation of extracellular signals, examples, mitogen-activated protein kinase (MAPKs). Mutations of K-raslead to a permanently active state that permits the cell to evade apoptosis and acquire a growth advantage.

    DEVELOPMENTAL PATHWAYS ALTERED IN COLORECTAL CANCER

    Wnt signaling :

    Signaling initiated by secreted glycoproteins of the Wnt family regulates many aspects of embryonic development and it is involved in homeostasis of adult tissues. In the gastrointestinal (GI) tract the Wnt pathway maintains the self-renewal capacity of epithelial stem cells (​Krausova & Korinek 2012​). In small intestine and colon, Wnt signaling controls the homeostasis of intestinal stem cells (ISCs) that fuel, via proliferation, upward movement of progeny cells from the crypt bottom toward the villus and differentiation into all cell types that constitute the intestine (​Avri Ben-Ze'ev, 2016​). Abnormal activation of Wnt signaling has been implicated in the regulation of pleothora of CSC types including colorectal cancer, breast cancer, hematologic effectors regulate various processes that are important for cancer progression, including tumor initiation, tumor growth, cell senescence, cell death, differentiation and metastasis (​Anastas & Moon 2013​).

    In the absence of Wnt ligands, the cytosolic β-catenin is phosphorylated for proteasome-dependent degradation by a " Destruction complex" consisting of axin, adenomatous polyposis coli (APC), glycogen synthase 3 β (GSK3 β), and casein kinase 1 α. However, in the presence of Wnt, signaling is activated through the ligands binding to the seven-trans membrane receptor Frizzled (FZD) and the single-membrane-spanning low density receptor-related protein 5/6 (LRP5/6). FZD then recruits the intracellular protein dishevelled (DVI), which subsequently sequesters Axin and GSK3 from the cytoplasm to the cellular membrane resulting in decomposition of the "Destruction complex". Consequently, the active unphosphorylated β-catenin accumulates and translocates into the nucleus to regulate target gene expression.

    WNT SIGNALLING FAISAL.jpg
      Wnt Signaling pathway

      Over activation of Wnt signaling is a hallmark of colorectal cancer (​Avri Ben-Ze'ev, 2016​​). The role of Wnt signaling in carcinogenesis has most prominently been described for colorectal cancer, but aberrant Wnt signaling is observed in many more cancer entities (​Zhan et al 2016​). The Wnt signaling pathway is a key regulator of both the early and the later, more invasive stages of CRC development. CIN (Chromosomal Instability pathway) is the most well characterized type of colorectal pathway and the most common. The tumorigenic process involves different mitotic spindle check point regulators and proteins that mutually influence mitotic chromosomal stability. A "Key"initial mutation is the early mutation of the adenomatous polyposis coli (APC) tumor suppressor gene. The APC tumor suppressor gene is involved in APC/β catenin. Its inactivation results in increased Wnt pathway signaling, through the failure to degrade β-catenin. The β-catenin cytoplasmic accumulation leads to its translocation into the nucleus and stimulates the TCF-targets, with increased proliferation, differentiation, migration and adhesion of colorectal cells (​Colussi et al 2013a​).

      HEDGEHOG PATHWAY:

      Hedgehog (Hh) pathway is a signaling cascade that directs patterning in most animals and is crucial for proper development (​Qian Zhao, 2012​). The Hedgehog family of secreted proteins governs a wide variety of processes during embryonic development and adult tissue homeostasis (​Chi-chung Hui, 2008​). Hedgehog is a morphogen that acts in a short or long range fashion on various tissue types. In mammals, there are three Hh proteins; Sonic Hh, Indian Hh, Desert Hh, out of which Sonic Hh is the best studied ligand of the vertebrate pathway. Hh signaling is most active during embryogenesis, and is mostly quiescent in adults but aberrant reactivation of the pathway in adult can lead to the development of cancer (​F. J. de Sauvage, 2006​). Therefore, recently it has been recognized as a novel therapeutic target in cancers. Basal cell carcinoma and medulloblastomas are two common cancers identified with mutations in components of the Hedgehog pathway (​Chang-Woo Lee, 2003​). The Hedgehog signaling pathway's vertebrates consists of the Patched receptor (PTCH) which is a 12-transmembraneprotein receptor and Smoothened (SMO, s 7-transmembrane protein related to G protein-coupled receptor) protein. There are also two PTCH genes, PTCH 1 and PTCH 2. All mammalian hedgehogs bind both receptors with equal affinity; hence PTCH 1 and PTCH 2 cannot distinguish between the ligands though both have a distinct downstream signaling activity. Downstream signaling of SMO in mammals is known as Glioma associated oncogene-GLI 1, GLI 2, and GLI 3. GLI1 appears to be soley an activator. It is believed that GLI2 and GLI3 are the primary transducers of Shh signaling, whereas GLI1, whose expression is transcriptionally regulated by GLI2 and GLI3, plays a secondary role in potentiating the Shh response (​Le Borgne 2005​).

      In the absence of Hedgehog ligand, PTCH located on the cell membrane at the base of primary cilia, cellular structure found in most mammalian cells, suppresses the SMO from entering the cilium, thereby preventing the initiation of downstream signaling events. PTCH acts like a sterol pump and removes oxysterols that have been created by 7-dehydrocholestrol reductase, thereby restraining the SMO initiated pathway. GLI 1 activators along with SUFU (Suppressor of fused) which is a negative suppressor prevents the transcription of GLI 1 target genes thereby keeping the pathway off.

      HEDGEHOG PATHWAY FAISAL.png
        HEDGEHOG PATHWAY

        INHIBITOR OF HEDGEHOG PATHWAY: CYCLOPAMINE

        Cyclopamine (11-deoxojerine) is a naturally occurring chemical that belongs in the family of steroid alkaloids. It is a teratogen from the corn lily (Veratrum californicum) that causes fatal birth defects. It prevents the fetal brain from separating into two lobes, which in turn causes the development of a single eye (Cyclopia). The chemical was named after this effect, as it was originally noted by Idaho lamb farmers who contacted the US Department of Agriculture after their flocks gave birth to cycloptic lambs in 1957. It then took more than a decade to identify the corn lily as the culprit. The poison interrupts the Sonic Hedgehog pathway during development, then causing birth defects.

        SANT-1 inhibits Sonic hedgehog signaling and induced apoptosis via Ras/NF-κB pathway in glioblastoma cells. SANT-1 is a potent sonic hedgehog pathway (Shh) antagonist that directly inhibits by binding to the smoothened (Smo) receptor. SANT-1 inhibits wild type and oncogenic Smo with equal potency.

        sant1.png
          SANT1 
          cyclopamine.gif
            CYCLOPAMINE

            METHODOLOGY

            Methods

            CELL CULTURE

            CELL LINE USED

            HT 29 : HT 29 is a human colon cancer cell line used extensively in biological and cancer research. HT-29 cells form a tight monolayer while exhibiting similarity to enterocytes from the small intestine.

            Media used was DMEM. PBS-EDTA were used for washing. Trypsin was used to detach cells.

            CULTURE MEDIA

            • DMEM powder- 1 packet
            • NaHCO3 - 3.7g
            • HEPES - 2.38g

            PBS-EDTA

            • NaCl -8g
            • KCl -0.201g
            • Na2HPO3 -0.61g
            • KH2PO4 -0.2g

            Add NaCl, KCl, Na2HPO4, KH2PO4 to 500ml distilled water and adjust the pH to 7.4, add 0.2g EDTA and make up the volume to 1L.

            A) CELL REVIVAL FROM CRYO

            MATERIALS REQUIRED

            • Frozen cells to be revived
            • complete growth medium, pre-warmed to 37º C
            • 70% ethanol
            • Tissue-culture flasks, plates or dishes

            PROTOCOL

            • Frozen cells were taken from -80ºC and thawed rapidly (<1 minute) in a 37º C water bath.
            • The thawed cells were diluted slowly, using pre-warmed growth medium.
            • The tubes were centrifuged at 130x g for 5 minutes.
            • The supernatant was discarded and the pellet was resuspended in 1ml media.
            • The media, containing cells were transferred into T25 flask.
            • 4 ml media was added.

            B) CELL COUNTING AND PASSAGING

            Passing also known as subculturing or splitting of cells involves transfer of a small number of cells into a new vessel. Cells can be cultured for a long time if they split regularly as it avoids senescence associated with prolonged high cell density.

            MATERIAL REQUIRED

            • DMEM with 10% FBS
            • PBS-EDTA
            • Trysin
            • 1x PBS
            • Hemocytometer
            • Culture flask

            PROTOCOL

            • The media was discarded and cells were washed with PBS-EDTA.
            • The cell monolayer was trypsinised by adding trypsin and keeping it in CO2 incubator for about 3-5 minutes. Media double the volume of trypsin was added to neutralize and centrifuged at 130g for 5 minutes.
            • The supernatant was discarded and the pellet was resuspended in media.
            • 10 times dilution was made (20μl cells + 180μl PBS) in another tube.
            • Cells were mixed gently.
            • Hemocyometer chamber and cover slip were cleaned with alcohol. Cover slip was dried and fixed in the position.
            • 15μl of diluted cells were added to the hemocytometer.
            • 20x objective lens was used to focus.
            • The cells were counted and calculated using the formula :

            Number of cells (x)= (total no of cells)/4 * 104

            Volume of cell suspension = required no of cells/X (per ml)

            The volume of the cell suspension to be taken from the quantified cell suspension is made up of the required concentration of cells per unit volume.

            C) TREATMENT OF CELLS BY SANT1 (Hh pathway inhibitor)

            The HT-29 cell line was plated in 60mm dishes and incubated for 48 hours at 37ºC , in 5% CO2 and 95% humidity. Then cells were exposed to SANT1 at 60nM and DMSO (0.04%) as control. Lastly, the cells were harvested and subjected to RNA extraction.

            ISOLATION OF RNA

            A) RNA ISOLATION BY TRIZOL METHOD

            MATERIAL REQUIRED

            • DMEM-FBS
            • PBS-EDTA
            • Trypsin
            • TRIZOL reagent
            • Chloroform
            • 100% ethanol
            • Nuclease free water

            PROTOCOL

            • Media was removed from the cells and washed with PBS-EDTA twice.
            • Cells were trypsined and centrifuged for 800g for 10 minutes.
            • Supernatnat was discarded and pellet was suspended in 1 ml TRIZOL reagent.
            • Incubated for 5 minutes.
            • 200μl chloroform was added.
            • The tube was vortex vigrously to make sure it is mixed properly.
            • Incubated at room temperature for 5 minutes.
            • Centrifuged at 12,000 RCF for 15 minutes at 4ºC.
            • Two layers were obtained. The aqueous layer was transferred to a new tube.
            • 0.66μl of glycoblue dye and 500μl of 100% isopropanol was added to the aqueous phase and mixed gently.
            • Incubate at -20ºC for 1.5 hours.
            • After the incubation, the tube was centrifuged at 12000x g for 10 minutes at 4ºC.
            • A glassy pellet was obtained.
            • The supernatant was removed and the pellet was washed with 1 ml 75% ethanol.
            • Centrifuged at 7500 RCF for 5 minuted at 4ºC.
            • The supernatant was discarded and the pellet was air dried.
            • The pellet was resuspended in nuclease free water and heated in heat block at 55ºC for 10 minutes.
            • The isolated RNA is quantified in Nano Drop-spectrometer.

            cDNA CONVERSION

            MATERIAL REQUIRED

            • Takara kit
            • PCR machine
            • RNA

            PROTOCOL

            • Primers and buffer were briefly vortexed.
            • Nuclease free water, buffer, oligo dT, RT enzyme and random primer were added to each tubes for making the master mix.
            • RNA samples were added to respective tubes and to that, master mix was added such that the final reaction voulme is 10μl
            • RNA was converted to cDNA using Pro Flex PCR system.
            • cDNA conversion occurs in one cycle, 3 stages -

            a) 37ºC (30 minutes)

            b) 85ºC (5 seconds)

            c) 4ºC ( infinity)

            cDNA CONVERSION (10μL reaction)

            • RT enzyme -0.5μl
            • Buffer -2μl
            • Oligo dT -0.5μl
            • Random primer -0.5μl
            • RNA -1ng
            • Nuclease free water -make upto 10μl

            REAL TIME PCR

            MATERIALS REQUIRED

            • Takara kit
            • PCR
            • 384 well plate

            PROTOCOL

            • The primers and buffer were vortexed and spinned.
            • Gene mix was prepared for each gene containing buffer, forward and reverse primers, SYBR green and nuclease free water.
            • PCR was carried out in 5 μl reaction mixture containing buffer, forward and reverse primers, SYBR green, nuclease free water and cDNA.
            • Reaction mix was loaded into 384 wellplate as triplicates.
            • The cDNA was amplified using 7900 HT FAST REAL TIME PCR.
            • The real time analysis were done by 2-∆∆Ct method using SDS 2.1 software.
            • Normalization of gene was done using GAPDH as endogenous control.

            PCR REACTION (5μl reaction)

            • SYBR green-2.5μl
            • Forward primer-0.1μl
            • Reverse primer-0.1μl
            • cDNA-0.25μl
            • Nuclease free water- upto 5μl

            ISOLATION OF PROTEIN BY RIPA BUFFER

            MATERIALS REQUIRED

            • 1x PBS
            • RIPA buffer
            • PHI
            • PI

            PROTOCOL

            • The plates were washed with 1 ml 1x PBS.
            • 1 ml 1x PBS was added and the cells were scraped out using a cell scraper.
            • The cells were collected into an eppendrof and centrifuged out using a cell scraper.
            • The supernatant was discarded.
            • 50-200μl of RIPA buffer was added based on the pellet size.
            • PHI and PI (10μl/1 ml buffer) were added.
            • The pellet was resuspended well and vortexed.
            • Mixed in thermomixer at 4ºC for 1.40 hours at 1400rpm.
            • Centrifuged at 140000rpm for 15 minutes.
            • The supernatant was collected and stored at -80ºC.

            PROTEIN ESTIMATION USING BRADFORD ASSAY

            Bradford asssay is a colorimetric assay, used to measure the total protein concentration in a sample. The principle of the assay is that the protein molecule binds to Coomassive dye under acidic condition which results in a color change from brown to blue.

            MATERIALS REQUIRED

            • BSA
            • MilliQ water
            • Bradford's reagent
            • 96 well plate

            PROTOCOL

            • BSA 1mg/ml stock was prepared.
            • Different concentrations (100, 50, 25, 12.5, 6.25 μg/ml) of standard were prepared from BSA stock solution by serially diluting it with milliQ water.
            • 50μl of standard solution BSA solution and 200μl Bradford reagent were added to the 96 well plates as triplicates.
            • 50μl distilled water and 200μl Bradford reagent serves as blank.
            • 50μl of test protein sample and 200 μl Bradford reagent was added as triplicates.
            • Absorbance was read at 595 nm using microplate reader.
            • The concentration of the unknown protein(x) was calculated using the formula

            X= (y-c)/m* dilution factor

            where, y-absorbance of unknown protein, m and c are the slope and intercept of

            the standard curve respectively.

            TREATMENT OF CELLS BY SANT1 (Hh pathway inhibitor)

            The HT-29 cell line was plated in 60mm dishes and incubated for 48 hours at 37ºC , in 5% CO2 and 95% humidity. Then cells were exposed to SANT1 at 60nM and DMSO (0.04%) as control. Lastly, the cells were harvested and subjected to protein extraction.

            SDS PAGE

            Sodium dodecyl sulphate polyacrylamide gel electrophoresis is the most widely used method for analyzing protein mixtures quanatitatively. SDS denatures the protein and imparts negative charge to it. Protein move towards the positive electrode. It is based on the separation of proteins according to their size. Smaller molecules elute faster the acrylamide mesh.

            MATERIALS REQUIRED

            • Glass plates
            • Acrylamide
            • SDS
            • APS
            • TEMED
            • Stacking gel
            • Separating gel
            • Electrode buffer
            • Isopropanol
            • SDS PAGE apparatus

            PROTOCOL

            • Glass plates and combs were washed and dried properly and wiped with 70% ethanol .
            • Glass plates were then assembled to make the gel molting cassette and checked for leak with distilled water.
            • Separating gel was prepared and poured in between the glass plates without bubbles.
            • Separating gel was overlaid with isopropanol and the gel was left to polymerize for 30-45 minutes.
            • Once the separating gel is polymerized, remove the isopropanol layer and add stacking gel without making air bubbles and quickly insert the comb. Allow the gel to polymerize.
            • After the gel is polymerized, the glass plates with gel were clamped to the electrophoresis chamber which was prefilled with 1x electrode buffer.
            • The comb was removed carefully and the wells were cleaned with electrode buffer.
            • The protein samples which were preheated at 95ºC for 5 minutes with loading dye containing β-mercaptoethanol and SDS, was loaded into the wells slowly.
            • The electrode were connected to the power cord. The power supply of 60V was given for the stacking gel and 100 V for the resolving gel at room temperature.
            • Once the tracking dye reaches the bottom of the gel, the glass plates were taken outside by disassembling the setup and the stacking gel was removed. Resolving gel was washed with transfer buffer.

            WESTERN BLOTTING

            Western blotting technique is used for identification of particular protein from the mixture of protein. In this method , labeled antibody against particular protein is used and the desired the protein is identified, so it is a specific test.

            MATERIAL REQUIRED

            • PVDF membrane
            • Fibrous pads
            • Whatman filer paper No.3
            • Towbins buffer
            • 1x TBST
            • Blocking solution
            • Secondary antibody
            • Forceps, scissors
            • Western transfer cassette and tank
            • Fixer, developer, water
            • X-ray film, X-ray film cassette

            PROTOCOL

            • PVDF membrane was fixed in methanol fo 15 seconds.
            • The resolved protein was transferred on to PVDF membrane by making a sandwich in a plastic transfer cassette.
            • The transfer sandwich was assembled in a tray filled with Towbins buffer in the order; fibrous pad-whatman filter paper-PVD membrane -gel-Whatman filter paper-fibrous pad.
            • The sandwich was assembled by gently pressing to avoid air bubbles.
            • The sandwich in the cassette was placed in a tank containing Towbins buffer. The leads of the power supply were connected to corresponding anode and cathode side of electro blotting apparatus.
            • A power of 100V was supplied for 2 hours for the transfer. The whole setup was covered with ice.
            • After the transfer, the membrane was blocked with 5% non-fat milk blocking solution for 1 hour and washed with 1x TBST three times at 5 minutes interval.
            • Incubated overnight in the primary antibody solution, aganist the target protein at 4ºC.
            • The blot was rinsed 3 times at an interval of 5 minutes with with 1x TBST on rocker.
            • After that incubation in the HRP-conjugated secondary antibody solution for 1 hour at room temperature.
            • The blot was rinsed 3 times at an interval of 5 minutes with 1x TBST on rocker.

            IMAGINING AND DATA ANALYSIS

            • Mix ECL solution 1 and 2 in 1:1 i.e luminol and H2O2.
            • The chemiluminescent signals were captured using a CCD camera based imager.
            • "Quantity one" software was used to read the band intensity of the target protein.

            RESULTS AND DISCUSSION

            Results

            CELL LINE USED : HT-29

            Morphology : HT-29 is a human colon cancer cell line used extensively in biological and cancer research. It is a human colorectal adenocarcinoma cell line with epithelial morphology. HT-29 cells form a tight monolayer while exhibiting similarity to enterocytes from the small intestine. Under standard culture conditions, these cells grow as a nonpolarized, undifferentiated multilayer. HT29 cells were grown in DMEM media with 10% FBS supplementation.

            ht 29 cell line.tif
              HT-29 Cell line 

              Culture: The HT-29 cell line was plated in 60mm dishes and incubated for 48 hours at 37ºC , in 5% CO2 and 95% humidity. Then cells were exposed to SANT1 at 60nM and DMSO (0.04%) as control.

              RNA ISOLATION, QUALITATIVE AND QUANTITATIVE ANALYSIS

              RNA QUANTIFICATION
              SAMPLECONCENTRATION (ng/μl)260/280260/230
              HT-29 DMSO 0.04%2071.51.882.01
              SANT1 60nM2246.31.831.89

              There were two samples, HT-29 DMSO control and treated with SANT1 (60nM).

              260/280 Ratio :The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indi-cate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.

              260/230 Ratio : This ratio is used as a secondary measure of nucleic acid purity. The 260/230 values for pure nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. If the ratio is appreciably lower than expected, it may indicate the presence of contaminats which absorb at 230nm.

              RNA QUALITATIVE CHECK:

              The overall quality of isolated RNA can be assessed by electrophoresis on a denaturing agarose gel. Thus, an agarose gel for checking the quality of RNA was run.

              RNA GEL.tif
                Agarose gel showing clear intact ribosomal RNA bands after isolation

                28S ribosomal RNA is the structural ribosomal RNA (rRNA) for the large component, or large subunit (LSU) of eukaryotic cytoplasmic ribosomes, and thus one of the basic components of all eukaryotic cells. It is the eukaryotic nuclear homologue of the prokaryotic 23S and mitochondrial 16S ribosomal RNAs.

                18S ribosomal RNA (abbreviated 18S rRNA) is a part of the ribosomal RNA. The S in 18S represents Svedberg units. 18S rRNA is a component of the small eukaryotic ribosomal subunit (40S). 18S rRNA is the structural RNA for the small component of eukaryotic cytoplasmic ribosomes, and thus one of the basic components of all eukaryotic cells.18S rRNA is the eukaryotic cytosolic homologue of 16S ribosomal RNA in prokaryotes and mitochondria.

                Because mRNA comprises only 1-3% of total RNA samples, it is not readily detectable even with the most sensitive of methods. Ribosomal RNA, on the other hand, makes up >80% of total RNA samples, with the majority of that comprised by the 28S and 18S rRNA species (in mammalian systems). mRNA quality has historically been assessed by electrophoresis of total RNA followed by staining with ethidium bromide. This method relies on the assumption that rRNA quality and quantity reflect that of the underlying mRNA population. Because mammalian 28S and 18S rRNAs are approximately 5 kb and 2 kb in size, the theoretical 28S:18S ratio is approximately 2.7:1; but a 2:1 ratio has long been considered the benchmark for intact RNA. While crisp 28S and 18S rRNA bands are indicative of intact RNA, it is less clear how these long-lived and abundant molecules actually reflect the quality of the underlying mRNA population, which turns over much more rapidly.

                GENE EXPRESSION ANALYSIS

                real 1.tif
                  SANT1 treatment effects the Hh signalling effectors along with cancer stem cell markers expression and drug transporters in HT29  cell line. 
                  oct4.tif
                    Relative expession of the cancer stem cell marker before and after treatment.

                    The relative mRNA expression of GLI1 and GLI2 (transcription factors) and ABCG2, ABCB2,ABCC1 (drug transpoters) and CD44, OCT4, NANOG (cancer stem cell markers) was reduced after the treatment of Hh pathway inhibitor; SANT1. The concentration of SANT1 has to be increased to further validate these results. Further studies and experiments are going on in this perspective.

                    WESTERN BLOT RESULT:

                    blot_1.tif
                      Relative expression of the cancer stem cell markers at protein level

                      The expression of CD133, CD44 after the treatment of SANT1 at 30nm and 60nm has no significant change when compared to the DMSO control. So, here also the concentration of SANT1 has to be increased. Beta actin is taken as an endogenous control. Futher studies and experiments are going on.

                      DISCUSSION

                      Colorectal carcinoma (CRC) represents the third most commonly diagnosed cancer in men and second in women, with more than 1.2 million new cancer cases and 600000 deaths only in 2008. Its prevalence is still on the rise in the developing countries due to the ageing population associated with a diet low in fruit and vegetables, but high in red meat, fat and processed food.

                      Two possible models of colorectal cancer carcinogenesis are currently described: a stochastic model, where any cell has an equal capacity of cancer initiation and promotion, and a cancer stem cell (CSC) model, where tumors are organized in a certain hierarchical degree and only CR-CSCs have a cancer potential. CRC carcinogenesis is a complex process requiring the accumulation of genetic/epigenetic aberrations.

                      Cancer stem cells (CSCs) play an important role in cancer development, because of their characteristics, which are their self-renewing capacity, chemoresistance, and their tumorigenic capacity ​Lin Chen, 2011​ . Several therapeutic strategies have been suggested to target CSCs.

                      Inhibiting the key signaling pathways that are active in CSCs is one of the most promising strategies for treatment of cancer. Hedgehog signaling (Shh) pathways are essential to regulate the self-renewal of CSCs and are aberrantly activated in a variety of cancers. Here we have used SANT1 as the inhibitor of Hh pathway. After inhibition, we were interested in investigating the role of Hh pathway in regulation of CD133, CD44, OCT4 expression (cancer stem cell markers).

                      CD133 and CD44 are two main markers that have recently been associated with CR-CSCs. Usually the expressions of these surface markers are relatively higher in cancer cells. Regarding the expression of CD133, several studies were performed using immunohistochemical methods showing that the CD133 antigen was located exclusively on the cell membrane at the luminal surface of cancer glands, while others demonstrated that CD133 could be detected both on membrane and cytoplasm in CRC (​Wang Q et al 2009​)

                      The cell adhesion molecule CD44 is a hyaluronic acid receptor which was proposed as an alternative CSC marker. CD44 is known to be involved in cell growth, differentiation and survival. As an important adhesion molecule, CD44 plays a major role in cancer cell migration being associated with tumor initiation in xenografts and colony formation, as well as tumor stage, lymph node infiltration, prognosis and survival.

                      Actually the over activation of the Hh signaling pathway is supposed to be one of the reasons in the onset of colorectal cancer. After inhibition we have analyzed the expression of the cancer stem cell markers.   We found that, at the working concentration of SANT1, both at the mRNA level and protein level, the expression of the markers were lowered but up to a significant value.So, for better results the concentration of the inhibitor has to be increased.

                      In conclusion overall, our experiments show that SANT1 has the potential to inhibit the Hh pathway and consequently decrease the expression of cancer stem cell markers but not very significantly. So, further studies are going on in order to find a better concentration of SANT1 such that it could inhibit Hh pathwayexpression of the surface markers.  

                      ACKNOWLEDGEMENTS

                      The satisfaction that accompanies the successful completion of any task would be incomplete without the mention of people who made it possible. I thank almighty for giving me the support and blessing to complete the project. First and foremost I would like to express my deep sense of gratitude to Indian Academy of Sciences (IASc-INSA) for providing me a golden opportunity to carry out this project.

                      I would to express my gratitude to Dr. Fahim Halim Khan, Assistant professor, Department of Biochemistry, AMU, Aligarh for recommending me to carry out this two month project.

                      I am grateful to prof. M. Radhakrishna Pillai, Director of Rajiv Gandhi Center for Biotechnology, for his critical supervision, advice, and constant encouragement.

                      I am deeply indebted to Dr. S. Asha Nair, Scientist F, Cancer Research Program, RGCB, for the invaluable help, stimulating suggestions and encouragement and helped me complete my work.

                      I am also grateful to Mr. Tapas Pradhan, phD student , RGCB, for his immense support, motivation and genuine encouragement throughout the period of my project which actually helped me to learn and improve.

                      I acknowledge Dr. Betsy. M, Mr. Samu John, Miss Anjana Soman, Ms. Ketakee Mahajan and Ms. Rajsree, who not only taught me the techniques but also refined my work professionly. It is also my pleasure to thank my lab mates Krishna R, Anna, Melvin, Irfan, Aavani, Anoopa, Niranjana, for their support and help. I would like to thank the technical staff of RGCB for their aid in smooth running my project.

                      Above all I am deeply indebted to my parents and my uncle Mr. Qasim Iqbal for their moral support and encouragement.

                      References

                      • R. Sever, J. S. Brugge, 2015, Signal Transduction in Cancer, Cold Spring Harbor Perspectives in Medicine, vol. 5, no. 4, pp. a006098-a006098

                      • Philip Martin, 2003, Notes

                      • Douglas Hanahan, Robert A. Weinberg, 2011, Hallmarks of Cancer: The Next Generation, Cell, vol. 144, no. 5, pp. 646-674

                      • Sam Al-Sohaily, Andrew Biankin, Rupert Leong, Maija Kohonen-Corish, Janindra Warusavitarne, 2012, Molecular pathways in colorectal cancer, Journal of Gastroenterology and Hepatology, vol. 27, no. 9, pp. 1423-1431

                      • Sam Al-Sohaily, Andrew Biankin, Rupert Leong, Maija Kohonen-Corish, Janindra Warusavitarne, 2012, Molecular pathways in colorectal cancer, Journal of Gastroenterology and Hepatology, vol. 27, no. 9, pp. 1423-1431

                      • Dora Colussi, Giovanni Brandi, Franco Bazzoli, Luigi Ricciardiello, 2013, Molecular Pathways Involved in Colorectal Cancer: Implications for Disease Behavior and Prevention, International Journal of Molecular Sciences, vol. 14, no. 8, pp. 16365-16385

                      • Hyun-Jin Shin, Kwan-Hyuck Baek, Ae-Hwa Jeon, Moon-Taek Park, Su-Jae Lee, Chang-Mo Kang, Hyun-Sook Lee, Seong-Ho Yoo, Doo-Hyun Chung, Young-Chul Sung, Frank McKeon, Chang-Woo Lee, 2003, Dual roles of human BubR1, a mitotic checkpoint kinase, in the monitoring of chromosomal instability, Cancer Cell, vol. 4, no. 6, pp. 483-497

                      • Dora Colussi, Giovanni Brandi, Franco Bazzoli, Luigi Ricciardiello, 2013, Molecular Pathways Involved in Colorectal Cancer: Implications for Disease Behavior and Prevention, International Journal of Molecular Sciences, vol. 14, no. 8, pp. 16365-16385

                      • Ali Mohammadi, Behzad Mansoori, Behzad Baradaran, 2016, The role of microRNAs in colorectal cancer, Biomedicine & Pharmacotherapy, vol. 84, pp. 705-713

                      • M. Krausova, V. Korinek, 2012, Signal transduction pathways participating in homeostasis and malignant transformation of the intestinal tissue, Neoplasma, vol. 59, no. 06, pp. 708-718

                      • Sayon Basu, Gal Haase, Avri Ben-Ze'ev, 2016, Wnt signaling in cancer stem cells and colon cancer metastasis, F1000Research, vol. 5, pp. 699

                      • Jamie N. Anastas, Randall T. Moon, 2013, WNT signalling pathways as therapeutic targets in cancer, Nature Reviews Cancer, vol. 13, no. 1, pp. 11-26

                      • T Zhan, N Rindtorff, M Boutros, 2016, Wnt signaling in cancer, Oncogene, vol. 36, no. 11, pp. 1461-1473

                      • Yong-Jun Liang, Qiu-Yu Wang, Ci-Xiang Zhou, Qian-Qian Yin, Ming He, Xiao-Ting Yu, Dan-Xia Cao, Guo-Qiang Chen, Jian-Rong He, Qian Zhao, 2012, MiR-124 targets Slug to regulate epithelial–mesenchymal transition and metastasis of breast cancer, Carcinogenesis, vol. 34, no. 3, pp. 713-722

                      • Jin Jiang, Chi-chung Hui, 2008, Hedgehog Signaling in Development and Cancer, Developmental Cell, vol. 15, no. 6, pp. 801-812

                      • M. Evangelista, H. Tian, F. J. de Sauvage, 2006, The Hedgehog Signaling Pathway in Cancer, Clinical Cancer Research, vol. 12, no. 20, pp. 5924-5928

                      • R. Le Borgne, 2005, The roles of receptor and ligand endocytosis in regulating Notch signaling, Development, vol. 132, no. 8, pp. 1751-1762

                      • Zhou Song, Wen Yue, Bo Wei, Ning Wang, Tao Li, Lidong Guan, Shuangshuang Shi, Quan Zeng, Xuetao Pei, Lin Chen, 2011, Sonic Hedgehog Pathway Is Essential for Maintenance of Cancer Stem-Like Cells in Human Gastric Cancer, PLoS ONE, vol. 6, no. 3, pp. e17687

                      • Qi Wang, Zhi-Guo Chen, Chang-Zheng Du, Hong-Wei Wang, Li Yan, Jin Gu, 2009, Cancer stem cell marker CD133+ tumour cells and clinical outcome in rectal cancer, Histopathology, vol. 55, no. 3, pp. 284-293

                      More
                      Written, reviewed, revised, proofed and published with