89 Figure 3.6 Molecular interaction of PKC-α with phytochemicals 90 Figure 3.7 Chemical structure of different alkyl cinnamates with their affiliation. 137 Figure 5.16 DNA fragmentation as evidence of apoptosis by drugs 138 Figure 5.17 DNA fragmentation in MDAMB-231 cells treated with different.
List of Tables
CGA Chlorogenic acid NADH Nicotinamide adenine dinucleotide DAG Diacylglycerol NFAT Nuclear factor of activated T cells. IP3 Inositol 1,4,5-trisphosphate Tris Tris(hydroxymethyl)aminomethane JAK Janus kinase VEGFR2 Vascular endothelial growth factor.
Units
Potentials of PKC in cancer development and therapeutic outcomes
Introduction
Maintaining a homeostatic environment in the body is achieved by an appropriate coordination between various cellular and organic systems. This homeostatic environment ensures the smooth functioning of all organs and organ systems that perform various functions in the body (Keesey and Powley, 2008).
Cancer facts and statistics
Cancer development and progression
The most important of these are chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections (Bosch et al., 2004). A low incidence (5 cases per 105) is found in women from countries such as Egypt, China and many European countries (Ferlay et al., 2010).
Mechanism of tumor formation
The global variation in cervical cancer rates is attributed to the spread of different subtypes of human papillomavirus (HPV), of which there are 15 high-risk human subtypes. Metastasis of cervical cancer is normally limited to adjacent organs around the cervix, such as the pelvis, rectum and bladder.
Different factors contributing to cancer development
- Environmental agents: The environment around a person decides whether he is being regularly exposed to toxic hazardous material as it play an important role in the development of cancer (Parsa,
- Hereditary factors: Development of cancer is linked to polymorphisms in tumor-suppressor genes, oncogenes, genes involved in the metabolism of endogenous or exogenous mutagens (Osborne
- Pathogens: During the later part of the 20th century, it was begun to be increasingly recognized the contribution of different pathogens in the induction of carcinogenesis in humans (Vandeven and
There is an increased risk of bladder cancer due to N-acetyltransferase 2 (NAT2) polymorphisms (Hein et al., 2000). Polymorphisms in the methylene-tetrahydrofolate reductase (MTHFR) gene are highly associated with colorectal cancer (Chen et al., 1999).
How cancer spreads to different parts of the body?
Breast cancer that has a hereditary family history is due to the prevalence of mutations in the BRCA1 or BRCA2 genes. Lymph nodes are present near every organ and organ system in the body.
Different therapeutic approaches to treat cancer
They interfere with the synthesis of purines and pyrimidines and block their synthesis (Longley et al., 2003). This process destroys the cell's ability to use its cytoskeleton in a flexible manner (Peltier et al., 2006).
Topoisomerase inhibitors: These drugs or their active metabolite cause the direct inhibition of topoisomerase I or topoisomerase II enzymes which are essential for DNA replication. This eventually
Chapter 1|17 Figure 1.9: Schematic diagram to explain the mechanism of action of paclitexel as an anticancer agent. This process prevents the DNA double helix from resealing, stopping the process of DNA replication.
Signal transduction inhibitors: Most of the new generation of drugs fall into this category. Some notable examples include cetuximab (Erbitux) which binds specifically to the extracellular domain of
Mitoxantrone and dactinomycin intercalate between base pairs via a non-intercalative electrostatic interaction, resulting in inhibition of DNA replication, RNA transcription, and protein synthesis (Figure 1.9). But instead of activation, this complex binds to these stretches of DNA elements as a repressor, where it recruits corepressors to shut down genes previously "turned on" by estrogen.
Epigenetic agents: These drugs are designed to effect chromatin modification of tumor suppressor genes or oncogenes. Vorinostat is known to inhibit the enzymatic activity of histone deacetylases
- Immunomodulatory agents: These agents boost the ability of the immune system to fight cancer
- Radiation therapy for cancer: Radiation therapy involves the use of high-energy radiation(X- rays, gamma rays, and charged particles) to shrink tumors and kill cancer cells (Klein and Dawson,
- Photodynamic therapy for cancer: Photodynamic therapy (PDT) for cancer takes advantage of substances called photosensitizers (PS) which can harvest the energy of a particular wavelength of
- Emergence of PKC as a master regulator in carcinogenesis
- Structural and Biochemical Details of PKC
- Targeting of PKC for anti-cancer drug development
- Aim and scope of the proposed work
- Identification of novel PKC-directed molecules from heterocyclic libraries, clinically approved drug-databank and phytochemical reservoir to develop potential anti-cancer drug: In
- Screening of identified molecules as potential ligands for PKC: After collecting the molecules, next, it was necessary to screen all the molecules, and to select only those that can serve as proper
- Validation and understanding the mechanism of action of PKC directed molecules in breast cancer cells: After selection of the best ligand molecules, it is time to explore the physiological
- To establish the potentials of PKC-directed molecules in anti-cancer drug-discovery programme: The final objective is to prove that the identified PKC-directed molecules have great
Several molecules such as PKC isozymes play a crucial role in the induction of carcinogenesis (Garg et al., 2014). Under stress (low oxygen conditions), PKC is activated in CAFs, which upregulates hypoxia-inducible factor-1 (HIF-1) (Lee et al., 2007).
The proposed study has following scope
Potentials of heterocyclic compounds, exploitation of drugs present in drug database as anti- cancer molecules: A vast repository of heterocyclic compounds exists in different chemical databases
An in-silico screening approach had to be chosen, which can effectively identify only those molecules that bind to the regulatory C1b domain of PKC with good efficiency. A clear understanding of the anti-cancer mechanism of action of PKC-targeted molecules together with a consideration of future experimental platforms will only strengthen the claims that these molecules should be taken up for drug discovery program.
To reveal the importance of natural molecules as modulators of PKC: The study also explores if popular beverages such as tea have the potential to act as adjuvants mainly as immune therapy for
Therefore, the present study shows the importance of heterocyclic compounds in anticancer drug treatment. 2) To reveal the importance of natural molecules as modulators of PKC: The study also investigates whether popular drinks such as tea have the potential to act as adjuvants. mainly as immunotherapy for.
Exploitation of clinically approved pharmaceutical drugs for cancer treatment: Another important aspect of this study is that it intends to uncover the potential use of drugs for other
- References
- Introduction
- Cell culture and treatments
- Cell viability assay
- Cell cycle analysis
- Acridine Orange & Propidium Iodide (PI) staining of apoptotic and dead cells
- DNA-fragmentation assay
- Intracellular ROS measurement
- Immuno-localization of PKC-α in MDAMB-231 cells
- Preparation of membrane and cytosolic fractions
- Immuno-blotting to detect PKC-α translocation
- Lactate Dehydrogenase Assay
- Immunoblotting to detect phospho-threonine proteins
- Measurement of change in mitochondrial membrane potential
- Immuno-localization to study cytochrome-c (cyt-c) release
- Caspase-3 assay
- Caspase-9 assay
- Estimation of lipid peroxidation level
- Estimation of protein carbonyl level
- Detection of 5'-nucleotidase in membrane fraction
- References
- Appendix I
- Appendix II
Further evidence that 3-phosphoinositide-dependent protein kinase-1 (PDK1) is required for the stability and phosphorylation of protein kinase C (PKC) isoforms. Activation of protein kinase Calpha inhibits growth of pancreatic cancer cells via p21(cip)-mediated G(1) arrest.
Hypotonic buffer (HEPES): 10 mM HEPES buffer pH 7.4
The resolving gel (Table 2.3) and the collecting gel (Table 2.4) were prepared using the ingredients described in Table 2.2. Finally, adjust the volume to two liters with MQ water Table 2.7: Recipe for the preparation of Transfer Buffer.
SDS-PAGE sample preparation and run: 36 µl of sample was mixed with 4 µl of 10x loading buffer and loaded onto 10% SDS-PAGE.
Protein estimation by Lowry’s method
Identification of PKC-directed ligands from different sources
Introduction
PKC signaling is modulated in cancer to modulate growth or apoptotic signals ( Castagna et al., 1982 ; Santiago-Walker et al., 2005 ). PKC acts as the downstream signaling molecule of many anticancer agents (Mackay and Twelves, 2003; Marengo et al., 2011).
Experimental procedures
- Collection of phytochemicals from medicinal plants found in north-eastern (NE) India: The list of plants present in north-eastern India with known medicinal properties was collected through
The results of competition docking experiments were taken into account for selection of the ligands. The toxicity and mutagenic potential of the top-hit heterocyclic molecules were predicted by ToxPredict (toxpredict.org/) (Willighagen et al., 2011).
Results
- Most of the top-hit heterocyclic compounds are non-toxic and non-carcinogenic: Toxicity &
- Top-hit heterocyclic compounds have many structurally similar drug counterparts available in the market: The top non-carcinogenic heterocyclic compounds identified were not available for further
- PKC directed molecules are found in phytochemicals from plants of north-eastern India
- Selected molecules bind strongly to PKC under in-silico conditions and form many interactions with the C1b domain of PKC: After the docking simulation was over, the PKC-ligand
HAMSTER ZINC00388658 Nemutageni ne-Mutageni ne-karcinogen ne-karcinogen ne-karcinogen Zinc11592646 Ne-matagenski ne-matagenski ne-makarcinogen ZIN-CARcinogen ZINCArcinogen ZINCArcinogeni CINC01420330, ki ne-Mutageni ZINCArcinogeni ZINC01420330 CINC0142036. -Carcinogen Zinc03872177 non-mutagen non-mutagen non-carcinogenic non-carcinogenic ZINC04102205 non-mutagen non-mutagen non-carcinogenic non-carcinogenic ZINC04215736 non-mutagen non-mutagenic non-carcinogenic non-carcinogenic ZINC05458886 non-mutagen non-mutagen non-carcinogenic non-carcinogenic no ZINC13298436 nemutageno nonmutageno non-mutagen non-mutagen non-mutagen ZINC14880002 nemutageno non-mutageno non-mutagen non-mutagen ZINC22026394 nemutageno non-mutageno non-mutagen non-rakotvorno non-rakotvorno Toksičnost in mutagene potenciale molekul svladiva je previdel server ToxPredict (.http://opentox.net/tutorials-category/toxpredict). All molecules bind to the polar ligand binding domain C1b PKC-α.
Discussion
Design, synthesis, molecular docking analyzes and in vitro ligand binding analyzes of these alkyl cinnamates by Mamidi et. These structurally different ligands use different parts of themselves to bind to the same site of the Cl domain.
Effect of Irbesartan on the development of diabetic nephropathy in patients with type 2 diabetes. Overexpression of protein kinase Calpha in MCF-10A human breast cells induces dramatic changes in morphology, proliferation, and motility.
Appendix I
Used as an astringent, diuretic and to treat gall bladder, dyspepsia and rectal cancer. Vitis vinifera European grape Used as an astringent, expectorant, diuretic, laxative and to treat cancer.
PKC-directed molecules affects breast cancer cells in multiple ways
Introduction
In Chapter 3, we discussed the research and identification of PKC targeting molecules from various sources. Thus, the current chapter describes experiments performed to understand the effects on breast cancer cells of treatment with selected PKC-targeting molecules.
Experimental procedures
So now that we have confirmed that the selected molecules bind strongly to PKC, it is very intriguing to ask the questions about how these molecules might influence the physiology of cancer cells. Few of the changes we are interested in are morphological changes, changes in the cell cycle and ultimately whether these molecules cause any change in viability (Figure 4.1).
Results
- PKC directed molecules induce morphological abnormalities in breast cancer cells: MDAMB- 231 and MCF-7 breast cancer cells were treated individually with different concentrations (0-100 μg/ml)
- Viability of breast cancer cells is compromised on treatment with PKC-directed molecules
MDAMB-231 or MCF-7 cells breast cancer cells were treated with various concentrations (0–100 μg/ml) of alkylcinnamon in serum-free media for 48 h. MDAMB-231 or MCF-7 breast cancer cells were treated with various concentrations of alkylcinnamon (0–100 μg/ml) in serum-free media for 48 h.
Discussion
Chapter 4|117 This change in cell morphology induced by PKC-directed molecules suggested a likely effect on the breast cancer cell cycle when treated with these molecules. Thus, in the current study, all selected PKC-targeting molecules induced cell death after induction of morphological abnormalities and cell cycle arrest in breast cancer cells.
The green tea catechin epigallocatechin gallate induces cell cycle arrest and shows potential synergism with cisplatin in bile duct cancer cells. Glycyrrhetinic acid induces G1 phase cell cycle arrest in human non-small cell lung cancer cells via the endoplasmic reticulum stress pathway.
PKC-directed molecules translocate PKC and induce apoptosis in breast cancer cells following mitochondrial
Introduction
In chapter 4, we have assessed that these molecules cause morphological abnormalities, dysregulate the cell cycle and cause cell death in breast cancer cells. We also do not know the mechanism of cell death caused by PKC-directed molecules in breast cancer cells.
Experimental Procedures
- Acridine Orange (AO) and Propidium Iodide (PI) staining: Propidium iodide (10 µg/ml) and acridine orange (1 µg/ml) at final concentrations were used to stain and distinguish live, apoptotic,
- Measurement of change in mitochondrial membrane potential: MDAMB-231 cells were seeded as ten thousand (10,000) cells per well of a 96 well plate in DMEM:F12 complete media for
After treatment, cells were prepared for lipid peroxidation level estimation as described in Chapter 2 (section 2.17). Then, cells were treated with different compounds (at their respective concentrations) for 24 h at 37°C in serum-free medium.
Results
- PKC-directed molecules translocate PKC-α from the cytosol to the plasma membrane in breast cancer cells: We decided to investigate if the selected PKC-directed molecules have the
- Danazol treatment causes downstream phosphorylation signal in MDAMB-231 cells
- Danazol abolishes tumorogenic (PMA) specific phosphorylation signal in MDAMB-231 Cells: We further asked whether Danazol has the potential to disrupt carcinogenic PMA specific
- PKC directed molecules induce degradation of genomic DNA during apoptosis of MDAMB-231 breast cancer cells: MDAMB-231 cells were treated individually with Danazol (70
- The apoptosis in breast cancer cells follow mitochondrial pathway
- PKC directed molecules cause protein carbonyl and lipid peroxidation in cancer cells
- Oxidative stress within breast cancer cells is responsible for apoptosis: We further explored the possible role of oxidative stress in the induction of apoptosis, cellular damage and death in cancer
MDAMB-231 cells stimulated with Danazol (70 µg/ml) yielded phosphorylated proteins in lysate, cytosolic and membrane fractions compared to cells stimulated with incomplete media (“untreated”). MDAMB-231 cells stimulated with PMA (100 ng/ml) yielded phosphorylated proteins in lysate, cytosolic, and membrane fractions compared with cells stimulated with serum-free medium ("untreated").
Discussion
However, there is always the possibility of extrinsic pathway involvement in caspase-3 activation (Figure 5.30) which remains to be explored. In the presence of N-acetyl cysteine (NAC), the level of oxidative stress in cells treated with the PKC-directed molecule was reduced (Figure 5.29-A).
Re-purposing of clinically approved drugs is an alternative to design anti-cancer drug: Drugs such as Danazol, Flunarizine and Cinnarizine that have been long used for the treatment of diseases
For example, we have seen that Danazol abolishes carcinogenic signaling induced by PMA, but we have not explored this function with other molecules. Although we are not sure if the similar mechanism is also followed by other molecules, it is very likely that they also follow that mechanism. But follow-up experiments with the other molecules are necessary to finally validate that these mechanisms are also followed well with other molecules.
Chemical synthesis of novel molecules targeting PKC can open up new avenues for anti-cancer drug development: Our study has also shown that the novel chemicals (alkyl cinnamates) synthesized
- References
Saurav Paul, Ashalata Roy, Suman Jyoti Deka, Subhankar Panda, Gopal N Srivastava, Vishal Trivedi and Debasis Manna. Protein kinase C (PKC)-mediated signaling is an essential pathway modulated by cancer cells to acquire a tumorigenic phenotype [ 2 ].
Alkyl Cinnamates Induce Protein Kinase C Translocation and Anticancer Activity against Breast Cancer Cells through Induction of the Mitochondrial
Cancer cells exhibit characteristics of unrestrained cell proliferation due to key alterations in several key signal transduction pathways [1]. Alkyl cinnamates induce protein kinase C translocation and anticancer activity against breast cancer cells through induction of mitochondria.
Accepted Article
Cytokines such as interferon-g are primarily responsible for overexpression of IDO1 enzyme in macrophages, epithelial cells and dendritic cells[7]. The overexpression of IDO1 enzyme is associated with poor prognosis in various cancers, including ovarian and pancreatic[1,3,8].
