Mutation family (H-ras, K-ras and N-ras) proteins with

Mutation in Ras and p53 genes is one of the major causes for many types of cancer and these are the most frequently mutated genes in human cancers.

Mutations in Ras and p53 genes may leads to change in structure and function of the gene products which results alterations in normal cell cycle and functions. The Ras family (H-ras, K-ras and N-ras) proteins with GTPase activity are the key components of different cell signaling pathways for cell proliferation and normal cell cycle. Variation in Ras function due to mutation results defects in Ras signaling and hyper proliferation of cells that may result in malignant transformation (1). Almost 30% of human cancers are known to have Ras mutations, but highest incidence is noticed in order: pancreas (90%), colon (50%), thyroid (50%), lung (30%) and melanoma (25%) cancers (2). p53 is a tumor suppressor protein encoded by TP53 gene.

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p53 plays a key role in cell cycle arrest and apoptosis following DNA damage. It is also called as the ‘the guardian of the genome’ or ‘the cellular gatekeeper of growth and division’. During cell division, p53 act as a checkpoint, where it finds DNA damage or mutations in the critical stages of cell DNA synthesis and mitotic phases of division.

In response to DNA repair systems, p53 stops cell division until the repair, before mutation pass through mitosis. If the damage of DNA is too high, p53 forwards these cells with mutation to apoptosis. Normally, p53 levels are kept at low by its negative regulator MDM2. MDM2 is responsible for proteasomal degradation of the p53 protein and also forms MDM2-p53 complex blocks p53 transactivation activity. In the conditions of DNA damage or oncogene activation, signals activate p53 for its transactivation activity to control cell growth, DNA repair and apoptosis in damaged cells. Mutation in p53 leads to uncontrolled growth of cells with mutant gene products may transform the cells to malignant or cancer.

Therefore cells that lack p53 or aberrant p53 pathway can pass mutations on to the progeny or next generation cells, which can induce tumor growth (3). About 50% of human cancers are associated with p53 mutations. Where, in other cases are associated with inactive p53 pathway in apoptosis or programmed cell death process.

About 60% of the lung carcinomas, 50% of colon carcinomas and 30% of breast carcinomas are with p53 mutations. So, the p53 based therapies got more importance in cancer treatment (4), which includes TP53 based gene therapy, p53 vaccination, small molecule activating p53 and inhibitors of MDM2 (5). A number of small molecules activating p53 via direct or indirect interaction have also reached the clinic. In these mainly advanced are the p53-MDM2 interaction inhibitors.

These agents can tightly bind to p53 and act as negative regulator to MDM2. Inhibition of MDM2-p53 interaction can stabilize p53 activity or functions and may propose a novel approach for cancer therapy. X-ray crystallography studies confirmed that the p53-MDM2 interaction at the amino termini of both proteins. Including p53-MDM2 interaction inhibition, these small molecules can also inhibit the expression of survival signals, cell cycle arrest and can increase p53 protein levels to induce p53 dependent apoptosis in cancer control (6,7,8). Most current chemotherapeutics follow the DNA damage response pathway to induce a genotoxic response in cancer cells. The increased DNA damage may leads to secondary malignancies. Hence, the combination of chemo preventive agents in cancer treatment is an attractive choice.

Target therapies without genotoxic burden on normal and tumor tissue is an alternative way to overcome these side effects in cancer treatment. Small molecules p53 activators are nongenotoxic nature and can work synergistically with other anticancer drugs. Combination therapies with different molecular mechanisms are considered more promising for higher effectiveness and also can work against multidrug resistant tumors. Many of dietary compounds plant flavonoids have been reported as cancer chemopreventive agents with anti-carcinogenic activity (9). These compounds also exert the anticancer activity through regulation of different cell signaling pathways include: regulation of Akt, NF-kB, COX-2 and different apoptosis pathways.

In this quercetin is most important dietary compound plant flavonoid, widely distributed in human diet and with chemo-preventive and anticancer properties. Quercetin is an excellent antioxidant capable of preventing oxidative stress and free radical formation. Along with antioxidant activity, quercetin also exerts several functions like cell growth signal control, block cell cycle at different phases and pro-apoptotic effect in wide varieties of cancer cells and animal models (10). Quercetin controlled tumor growth via different cell signaling pathways and apoptosis pathways have been well documented. Quercetin control drug induced genotoxicity in normal and cancer tissue and its antiproliferative effects has been reported in culture tumor cells of the human breast (11), prostate (12), squamous carcinoma-tongue (13) gastric cancer (14). Immunocytochemical studies demonstrated lower concentrations of quercetin inhibits p21-RAS protein expression and its GTPase activity in human colon cancer cells and primary colorectal tumors (15).

Quercetin induce antiproliferative effects from the modulation of estrogen-receptor mediated signal transduction pathways and also demonstrates synergy and reversal of the multidrug resistance phenotype when combined with chemotherapeutic drugs, in vitro. Quercetin in combination with cisplatin works synergistically and amplified anti proliferative and proapoptotic effects, dose and duration depended manner in small cell lung cancer cells (16). Quercetin protective role against cisplatin induced clastogenicity and apoptosis have been reported in mice bone marrow cells (17). Quercetin can alter numerous signal transduction pathways involved in cell division and programmed cell death. Quercetin-induced cell death studied by investigating the expression of Bcl-2 family proteins and caspases in HeLa cells (18, 19). The aim of the present study is to evaluate the combination effect of natural compound quercetin and its anticancer properties in synergism with the selected small molecule p53 activators works via inhibiting p53-MDM2 interaction. In this assay, natural compound quercetin and small molecule p53 activator (p53-MDM2 inhibitor) are selected for testing their combination anticancer effects in different cancer cells.

Anticancer activity of natural compound in combination with selected p53 activator via targeting Ras and p53 mediated apoptosis pathways in cancer treatment has not been tested so far. Also the selected pathways are with complex signaling network with multiple signaling molecules, activators and effectors which regulates cell proliferation and apoptosis. After detailed analysis of p53 pathway and role in apoptosis, p53 based cancer therapy got more importance and extensive research is in progress. p53-MDM2 interaction inhibitors are most advanced in the development of powerful drug combinations that may increase the selective chemotherapy and protection of normal cells and tissue. Including p53 based cancer therapies, selected p53 activator effect on other cell signaling pathways are still required to investigate. Role of plant flavonoids in p53 based cancer therapies has still need to investigate due to their wide distribution and normally present in regular human diet. Promising evidence suggest that chemopreventive agents might be combined with chemotherapy for the more effective treatment of cancer. These combinations can enhance the efficacy of chemotherapies and selected p53 based target therapies by modifying the activity of key cell proliferation, survival pathways and apoptosis process in cancer cells.

The objective of present research plan is to determine the synergistic anticancer activity of novel combination of natural compound quercetin with the selected p53 via p53-MDM2 complex inhibition. Rationale for selecting the suitable p53 activator is as follows: • drugs targeting p53 negative regulators and stabilising p53 functions for apoptosis pathway exhibit significant anti-cancer effect with less genotoxicity • Drugs targeting both AKT pathway and p53 mediated apoptosis are more effective • Inhibition of antiapoptotic proteins, cell growth signals and upregulation of proapoptotic members. In this synergism, quercetin and selected p53 activator (p53-MDM2 inhibitor) control tumor growth by negative regulation of different cancer cell growth signals and stabilising p53 activity in apoptosis process.

This synergism regulates the cancer cell growth in different ways: by controlling cell growth signals, and induce the apoptosis by upregulation of proapoptotic members and increase p53 activity. This may results in an increase rate apoptosis in cancer cells and become a novel combination in cancer treatment without any significant side effects in normal cells. Methodology (Materials and Methods):Evaluation of Anticancer Activity Cell Lines: The human cancer cells HCT-116 (Colon), A-549 or NCI-H460 (Lung), MCF-7 (Breast) and HeLa (Cervical) will be cultured in a suitable medium supplemented with serum and antibiotics (100 IU penicillin, 50µg streptomycin/ml) in a humidified CO2 incubator at 37 oC and 5% CO2 (20,21). Cultured cell lines will be treated with selected compounds and the anticancer properties will be evaluated by the following assays. Cell Viability Assay: MTT cell viability assay will be followed to determine the natural compound and p53 activator mediated inhibition of cell proliferation in different types of cancer cells. Test drugs will be applied individually and in combination. The IC (Inhibitory concentration) value will be calculated by probit analysis – GraphPad prism software. The percent cell viability and inhibition will be calculated using the following equation.

% Cell Viability= OD Test/OD Control x 100% Inhibition of proliferation= % Cell viability of control – % Cell viability of drug treated Colony formation assay: Selected drugs induced cytotoxicity or effect on clonogenicity will be determined by following colony formation assays. Colony forming assays are superior indicators of drug- induced cytotoxicity and effects on tumor cell clonogenicity than the regular cell viability assays. Cultured cells will be treated with selected drugs individually and also with their combinations. Following the treatment, cells will be trypsinised and counted by trypan blue dye exclusion assay. Approximately, 100 cells for each drug concentration or control will be seeded in cell culture plates and incubated for another 2 -3 weeks.

After incubation period colonies will be counted and the drug treatment effect will be determined as percentage inhibition of colony formation and was calculated using following formula; Cell migration assay: Cell migration assay and also called as scratch assay is an easy and well-developed method in vitro to evaluate the effect of drugs on cell-matrix and cell-cell interactions on cell migration, which mimic cell migration in vivo, general occurs during embryogenesis or tissue regeneration or cancer metastasis. Cultured cells will be treated with selected drugs and individually and in combination for 48 hours. After treatment a scratch will be created with a sterile p200 pipette tip and the floating cells will be removed.

Cell migration or closing of gap due to the migration of cells will be observed at different intervals using an inverted phase microscope and the images will be captured and the scratch area will be measured with Wimasis image analysis software.Selected drugs induced reduction in the % cell migration at each time interval compared to control using the following formula. Combination effect analysis: The effects of selected drugs on cell viability, colony formation and cell migration in different cancer cells were further analyzed to decide the drug interactions for synergy or additive effects based on the combination index (CI) value (Chou-Talalay method), calculated using CompuSyn software.

CI value is 1, it is ‘antagonistic’ (22,23,24). Combination effect on cell cycle analysis – Flow cytometry: The cell cycle analysis of selected human cancer cells will be performed by flow cytometry to estimate the cells distribution in different phases of cell cycle. Cultured cells will be treated with alone, and in combination of drugs and after drug treatment, the cells were harvested, washed and prepared for flow cytometry to determine the percentage distribution of cell cycle phases for untreated and drug treated groups of each cell line. Apoptosis Assay: The apoptosis in selected cancer cells induced by natural compound and p53 activator in combination and individually at different combinations will be determined by using the following methods: DAPI staining for fluorescent microscopic examination of apoptotic cells:Cultured cells will be treated with both the drugs alone, and in combination for 48 h. After treatment cells will be fixed and stained with DAPI stain (1 mg/ml) and then a drop of cell suspension will be placed on a slide and mounted with cover slip to examine under fluorescent microspore.

The slides will be screened for the quantification of cells showing features of apoptosis, and the results will be expressed as % apoptotic cells. Estimation of caspases-3 and -9 activity:The activity of caspases-3 and -9 will be measured using colorimetric assay kit from commercial supplier and the procedures will be followed as per the manufacturer instructions. Western Blot Analysis: Cultured cancer cells will be treated with selected drugs, individually and in combination. Expression of different proteins of Ras and PI3K/Akt pathway and key apoptotic proteins (proapoptotic, antiapoptotic, p53 and p53 inhibitors) will be determined by performing Western Blot analysis or specific protein determination methods like ELISA.

Statistical Analysis: The statistical significance of differential findings between treated and control will be determined by ANOVA (comparing mean difference between the groups), Dunnett’s t-test (pair-wise comparison) and regression analysis (dose response) using software, GraphPad prism, version 6.01. When calculated, p values smaller than 0.05 will be measured as statistically significant.

Results: It is anticipated that the studies proposed using combination of natural compound and p53 base therapy will make out very effective/optimal ratios of the compounds that lead to synergistic inhibition of selected cancer cells growth. This synergism controls the cancer cell growth by modulating different key survival signals and may induce apoptosis by stabilising p53 structure and functions. The results may show significant difference in cancer cells growth in combination than individual. Selected natural compound may also show genoprotective role and play significant role in cancer prevention.

Outcome and Significance:Completion of the proposed research plan is anticipated to yield information on the synergistic anticancer activity of potential combinations of natural compound and selected p53 activator that can be evaluated in designing future clinical trials for cancer treatment. Chemopreventive nature of natural compound quercetin in p53 based cancer therapies may explain the role of plant flavonoids in cancer prevention and synergistic mode of action in combination therapies. Pilot study:Palamur Biosciences Pvt. Ltd. conducted studies on antiproliferative activity of quercetin against different colon cancer cells. Results explained that quercetin works effectively against selected colon cancer cells alone and in combinations.

Funding:This project was approved for funding from the Department of Science ; Technology, Government of India under KIRAN DIVISION. Project title: Evaluation of synergistic anti-cancer Properties of quercetin and p53 Activators (both wild and mutant p53 binders) in colorectal cancer.