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Effect of Hyperbaric Oxygen Treatment on Methylome Proposal

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Analysis of Hyperbaric Oxygen therapy’s effect on Methylome of Cervical cancer cells by using Next Generation Sequencing techniques and quantifying Transcriptome using RT PCR

D.PROJECT DIGEST. Describe the proposed research geared to the non-specialist reader (max. 250 words).

Cancer cells are fast growing entities so they need more nutrients and oxygen etc. But in fast growing tumor mass there is oxygen depletion called hypoxia. Normal cells die in hypoxic condition but cancer cells get adapted to it by making changes in their metabolism. So considering this it is established that if we provide pure oxygen at high pressure it kills cancerous cells because they have been adapted to low oxygen. This high pressure oxygen treatment is called HYPERBARIC OXYGEN (HBO) therapy. HBO had showed effects in controlling cancer even in malignant form. But there is a debate going on about the safety of this method as some people believe it increases the cancer growth and doesn’t give results in cervical and urinary bladder cancers. In order to get insight into this problem we shall check the differential gene expression in cervical cancer cells after exposing to HBO treatment. It will tell us exact molecular basis of treatment’s effect on the genes. This is important because in cancer cells oncogenes (cancer causing genes) are overexpressed while tumor suppressor genes got silenced. Normally oncogenes are silenced because of methyl group attached on their nucleotides, called methylation. So by studying the methylation status of whole genome of cancerous cells after giving HBO treatment we’ll come to know either this treatment is supporting oncogenes or tumor suppressor genes in cervical cancer cell. Moreover we’ll get some idea why cervical cancer is not giving response to this treatment.

Project Details


Describe the proposed research. (max. 100 words)

In this study we’ll analyze the methylation status of whole genome causing differential gene expression of oncogenes and tumor suppressor genes in cervical cancer. We’ll perform this experiment both in vitro on cell lines and in vivo on mice with induced cervical cancer. Cells and mice will be exposed to high oxygen pressure in pure form at 3a.t.m for different intervals and then check their DNA by high throughput sequencing (including bisulphite technique) along with transcriptome level with RT PCR. Then sequence will be compared with normal cell’s DNA and cervical cancer cell DNA (without HBO treatment), it will give us the differential methylation pattern and gene expression in all three categories of cells i.e. cancer cells/mice with cervical cancer treated with HBO, normal healthy cells from mice cervics and untreated cervical cancer cells. Then it’ll be analyzed the effect of treatment on genome level.



Hyperbaric oxygen treatment affects Methylome of cervical cancer leading to differential gene expression. By checking this it’ll give us an idea about efficacy and safety of this treatment.


  1. To get an understanding how hyperoxic environment effect on cancer cells at genome level.
  2. Visualization of differential methylation pattern in response to HBO treatment.
  3. To understand the impact of methylation status of promoter, enhancer and body of the gene on the cellular response to change in external environment.
  4. It will give an idea of cancer cell adaptations to low oxygen by modifying their metabolism due to changes in their gene expression.
  5. To validate the effectiveness of HBO therapy because it is important in cancer treatment having minimum chances of side effects a compare to Chemo.
  6. Searching new genes involve in tumor suppression or tumor progression.


The introduction should consist of three paragraphs; the first paragraph should indicate the scientific hypothesis/commercial basis on which the project is based. The second paragraph should introduce the precise nature of the project, and the final paragraph should indicate the proposed objectives in the light of the first two paragraphs and explain clearly what the reader will see in the main body of the proposal.

Cervical cancer arises from uncontrolled division of cervical squamous cells. It has the ability to grow and invade to other parts of the body via blood stream. Its initial signs include cervical pain, bleeding from vagina and miscarriage (National cancer institute at national institute of health). Worldwide it is at fourth common cancer type in women and leading cause of women deaths due to cancer after breast and lung cancers (World cancer report 2014). In 2012 there were 528,000 cases reported and out of these 260,000 women died worldwide (Cancer research UK). It is about 8% of total deaths in the world due to cancer and 70% of cervical cancer patients reported in developing countries including Pakistan (World cancer report 2014). So this data tells us how important is to find efficient ways of cervical treatment. There are different treatment methods are being used today i.e. Chemo, radiotherapy, surgery and least common is Hyperbaric oxygen (HBO) therapy (Cervical cancer treatment at national at NIH, USA). HBO provide increased levels of extracellular Oxygen independent of Hemoglobin transport system (Gill AL, Bell CAN, 2004 Hyperbaric oxygen: its uses, mechanisms of action and outcomes). This high level of oxygen penetrates into cancer cells and results in their death due to activation of apoptotic pathway in cancer cells (Chen et al & Kawasoe Y et al). Most of the studies on HBO treatment are based on clinical observations of cancer mass response regulation of cellular metabolic and growth factors. There should be some genome wide study on methylation status of Oncogenes or tumor suppressor genes in cancer cells especially on cervical cancer as it showed least response to this treatment in clinical trials (Daruwalla et al 2004). So this research will fill the gap between cellular response and gene expression in HBO therapy.

This project will cover the epigenetic and transcriptomic aspects of Hyperbaric Oxygen treatment. It will provide us the clear picture that how cancer cells response to hyperoxic environment by manifesting their response in form of Differential gene expression. It is based on the post genomic study of methylation’s role in the cancer. As it well established that normally oncogenes are suppressed due to hypermethylation and tumor suppressor genes are active due to hypomethylation (Determining the Effect of DNA Methylation on Gene Expression in Cancer Cells by Chai-Jin Lee). Cancer occurs when scenario reverses due to any carcinogenic effect. So any treatment which affects cancer cells it may cause some changes in their Methylome. This project will cover the aspects of changes in methylation pattern of whole genome and transcriptome in response to HBO therapy.

Once this project gets completed we’ll able to link the effect of HBO treatment to the gene expression at cellular level. This will be helpful in finding new genes or pathways which are responsible for cervical cancer. This epigenetic study will provide the basis of cellular response to hyperoxic environment at epigenetic level and may provide new targets for cancer treatment. This research will solid evidences about the efficiency and safety level of HBO treatment and disclose the reasons of less response of cervical cancer to it. This will provide future prospects of modifying this technique to get much better results even in cervical cancer. Moreover it will tell about methylation changes in cancer cells before and after treatment which would be helpful in understanding the epigenetic basis of cancer occurrence.


A comprehensive and up-to-date literature survey clearly highlighting the existing gaps and what new information will be added to the existing pool of knowledge.

Cancer cells grow very fast that’s why they need more oxygen and other cellular nutrients as compare to normal cells. But cancer cells face a problem of efficient oxygen supply in solid tumor masses leading to almost 0 mmHG pO2 at center of tumor (H. W. Puffer et al). Tumor cells try to compensate this different ways like by making changes in their metabolic system. Normal cell get energy from mitochondrial respiration but tumor cells starts aerobic glycolysis or fermentation, this phenomenon is called Warburg effect (Matthew G. Vander Heiden). After getting hypoxic shock tumor cell turn on three types of processes; 1) angiogenesis (formation of new blood vessels), 2) deregulation of apoptotic pathways, 3) glycolysis shift (Darruwala et al). All these modifications are made to survive cancer cells and to adapt in low oxygen supply. Angiogenesis starts after expression of HIF 1 & HIF 2 as result of hypoxic shock. HIF 1 causes expression of VEGF (vasicular endothelial growth factor) which is involved in angiogenesis (Horiuchi A, Imai T, Shimizu M, et al). Moreover FGF (fibroblast growth factor) and angiogenin also produced as result of hypoxic shock leading to angiogenesis in the tumor (Kuwabara, et al). Apoptosis is blocked by different ways. One is by deregulating p53 which is a strong tumor suppressing factor (Jack T. Zilfou et al). Other includes hyper activation of Bcl2 and down regulation of Bax gene results in blocked apoptotic pathway (Hockenbery.D et al). Glycolysis shift occurs due to decrease in oxygen leves in tumor cells leading to increase in production of Glycolysis enzymes GLUT 1 & GLUT 3 along with decrease in enzymes involved in oxidative phosphorylation (Kondo Y et al).

Hyperbaric oxygen (HBO) treatment is being used from many years for wound healing and epithelialization. Based on the above mentioned discoveries that cancer cells are adapted to low oxygen, its use was expanded to cancer treatment thinking that high oxygen pressure might kill tumor cells. But it brought certain conflicts as well. This treatment proved fruitful in controlling many cancers but as oxygen is necessary for angiogenesis and cell survival, it is proposed that it can aggravate cancer instead of curing. In early 2000 Darruwala et al and Feilder et al took this matter seriously and proved that HBO does not promote cancer. After that many clinical trials and researches approved its effectiveness in many types of cancer. HBO treatment reduces mammary gland tumor (Raa A et al), Osteosarcoma (Granowitz et al), nasopharyngeal carcinoma (Peng ZR et al), Prostate cancer (Chong et al), colorectal cancer but in combination with radiotherapy (Dische & Senanayake). Now a days HBO is being used as an adjuvant with conventional cancer therapeutics like Chemo and radio therapy. Hypoxia hinders the chemotherapeutic treatment but HBO reduces this resistance of cancer cells by increasing cellular perfusion and sensitivity to Chemo (Al waili et al). Apart of all these effectiveness HBO did not show improvement in Cervical cancer in clinical trials (Darruwala et al). This project is designed to unveil the factors which make the cervical cancer unresponsive to HBO therapy while other cancer types are sensitive to this. Moreover there is gap between clinical results of this treatment and genetic or epigenetic regulations, in the literature. All of the previous studies are based on clinical manifestations of tumor progression, repression or cellular signaling responses to HBO treatment. So there is a need to link the clinical results to Genome wide regulation of methylation status of genes especially oncogenes and tumor suppressor genes.

In order to study the Methylation in differentially expressed genes we’ll use next generation sequencing techniques combining with bisulphite treatment and compare the methylation in normal cells, cervical cancer untreated cells, cervical cancer cell line treated with HBO. Moreover mice with induced cervical cancer will also be treated and the effect of HBO on tumor growth will be checked along with sequencing of their cervical cells too. There are differentially methylated regions are present in the genome, normally in the promoter and genic regions but intergenic regions may also be methylated. Aberrant methylation status in regions of genome like oncogenes or tumor suppressor genes will lead to cancer (Chai-Jin Lee et al, 2013). Bisulfite treatment technique is most commonly being used to study methylation of DNA. It was first introduced by Hyatsu et al in 1970s. This treatment converts unmethylated Cytosines into Uracil and 5mC remain unchanged which is then PCR amplified and sequenced, this sequence is compared with reference sequence to see the exact number of methylated cytosines (Clark, Horrison et al 1994).

  1. Now a days bisulfite conversion method in combined to next generation sequencing techniques called BS-Seq (Felix Krueger et al). Other advanced methods are also present like MethylCapseq in which methylated DNA is purified by affinity followed by next generation sequencing (Chai-jin lee et al). Another technique making use of bisulfite conversion is COBRA developed by Zhenggang et al in 1997. It is based on restriction digestion of methylation dependent sequences after bisulfite treatment. Another microarray based technique is MIRA-assisted microarray analyses in methylated DNA binding domain are being used to isolate methylated DNA regions followed by sequencing (Tibor Rauch et al 2006).

All of these techniques has same objective which is find methylation status of the genome and correlate this to the expression of gene. Gene expressions can be measured by RT PCR which quantifies the transcriptome of the cell. After the accomplishment of this project we’ll be able to link the Hyperbaric oxygen treatment on cervical cancer cell to the methylation differentiation in their genome.



  • Cervical cancer cell lines will be obtained from ATCC and culture in recommended condition.
  • On the other hand 5 Mice of 2 month age will be rasied with induced cervical cancer by locally injecting HPV in the cervics.
  • Next we’ll do HBO treatment with 100% pure oxygen at 3a.t.m for 10 to 12 exposers of 30min/day to both cell line and mice.
  • After that we will extract tissue sample from mice and cell from cultures and DNA will be extracted with QIAamp DNA mini kit and methylation will analyzed by Epigenome/TruSeq DNA methylation kit of Illumina.
  • Transcriptomic analysis will be done by extracting RNA using Illumina total prep RNA amplification kit and analyzing by Illumina sentrix human 6 version 2.
  • Methylation status of untreated cancer cells and normal cervical cells will also be obtained will be used as reference sequence.


Phase 1: Duration 2 months

In phase cells will be ordered, mice will be raised and tumor will be induced in their cervics.

Phase 2: Duration 6 months

Plan will be executed as mentioned in proposal.

Phase 3: Duration 5 months

Research data will be compiled and sent for getting published.


  • Oxygen Levels in Tumors in C3H Mice During Hyperthermia: Initial Data (H. W. Puffer)
  • Hypoxia-induced changes in the expression of VEGF, HIF-1 alpha and cell cycle-related molecules in ovarian cancer cells (Horiuchi A, Imai T, Shimizu M, et al.)
  • Hypoxia-mediated induction of acidic/basic fibroblast growth factor and platelet-derived growth factor in mononuclear phagocytes stimulates growth of hypoxic endothelial cells (Kuwabara, et al).
  • Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. (Hockenbery.D et al)
  • Hyperoxia retards growth and induces apoptosis and loss of glands and blood vessels in DMBA-induced rat mammary tumors, (Raa A et al).
  • Determining the Effect of DNA Methylation on Gene Expression in Cancer Cells ,Chai-(Jin Lee).
  • MIRA-Assisted Microarray Analysis, a New Technology for the Determination of DNA Methylation Patterns, Identifies Frequent Methylation of Homeodomain-Containing Genes in Lung Cancer Cells, (Tibor Rauch).
  • Deep sequencing reveals distinct patterns of DNA methylation in prostate cancer (Jung H. Kim)
  • Enrichment-based DNA methylation analysis using next-generation sequencing: sample exclusion, estimating changes in global methylation, and the contribution of replicate lanes. (Trimarchi MP).
  • Hypoxic Cervical Cancers with Low Apoptotic Index Are Highly Aggressive (Michael Höckel, Karlheinz Schlenger, Susanne Höckel, et al).
  • Detection of Epidermal Growth Factor Receptor in the Serum of Patients with Cervical Carcinoma, (Min-Jeong Oh).
  • Phase II trial of radiotherapy after hyperbaric oxygenation with chemotherapy for high-grade gliomas, (K Ogawa).
  • Hyperbaric oxygen: its uses, mechanisms of action and outcomes, (A.L.Gill,C.N.A.Bell).
  • Hyperbaric Oxygen Therapy for Malignancy: A Review (Jurstine Daruwalla, B.Sc, Chris Christophi, MD) 
  • Ozone Therapy and Hyperbaric Oxygen Treatment in Lung Injury in Septic Rats (Levent Yamanel)
  • Hyperbaric-Oxygen Therapy, (Patrick M. Tibbles, M.D).
  • Oxygen distribution in squamous cell carcinoma metastases and its relationship to outcome of radiation therapy, (Robert A. Gatenby,M.D).


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