Chemotherapy of Cancer Cell Lines Using Inflammasome Stimulation and Mitochondrial Cell Death Signaling Chara Charsou

MSc programme in Clinical Pharmacology and Therapeutics Democritus University of Thrace Bogazici University 14 February 2011

Cancer

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In the developed world 1 every 3 people will develop cancer during their lifetime



In 2004 7,4 million deaths worldwide



Disease of uncontrolled cell proliferation/dysfunction of apoptotic mechanism

Cancer is a genetic disease 

Environment (obesity, tobacco exposure, alcohol, air pollution, viral infections)

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Genetic profile major role. alterations in - tumor suppressor genes (BRCA, TP53) - proto oncogenes (bcl-2 family, Ras) - DNA repair genes - micro RNA genes

Chronic inflammation caused by bacterial infections (Streptococcus bovi, Chlamydia pneumonia) (Vogelman et al., 2007, Samaras et al., 2010)

Breast cancer 

Most common malignancy in women



95 per cent of cases sporadic/ 5 per cent inherited

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Mutations in TP53, BRCA, NFκB, CAN and ER related genes. Inflammatory breast cancer is characterized by NFκB target overexpression poor prognosis and survival rate < 35-40 per cent Anthracycline systemic therapy poor results (Robertson et. al. 2010)

Colon cancer 

One of the most common malignances, in the top 5 of cancer worldwide



Two types of colon cancer, sporadic and inherited (Family Polyposis, Hamartomatous Polyposis and Hereditary Nonpolyposis Colorectal Cancer)

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Genetic and chromosomal instability, mutations in TP53, Wnt pathway and the β-catenin protein inhibitor, Caspase 5 and Bax, genes controlling cell cycle, TGF-β and RAS-MAP pathways inactivation and activation respectively (Laurent-Puig P et al. 2010)



(anti-EGFR) monoclonal antibodies cetuximab and panitumumab new therapeutic options for metastatic colorectal cancer (mCRC) (A Lie`vre et al.2010)

Hepatocellular carcinoma  

 

 

Serious liver malignancy Caused mainly by HVC / HVB abuse

chronic inflammation and alcohol

TGF-β, Jak/STAT pathway and genes controlling cell cycle found to be downregulated Wnt pathway, SGK, STAT1 and many others found to be upregulated. (Maass et al., 2010) Many treatment approaches transplantation, percutaneous ethanol injection, radio waves, chemotherapy Most promising the VEGF and EGFR inhibitors Bevacizumab and Sorafenib (Llovet JM et al 2008)

Melanoma 

Originates from malignant transformation of melanocytes



Although not common skin cancer is the most lethal.



Extended exposure to the sun causes various mutations found in melanoma, such as B-Raf , K- Ras and other compounds of the Raf- MEK-ERK pathway, CDKN2A, CD 40, Apaf-1 and others (Soengas et al, 2001 )



Therapeutic approaches except from surgery are IL-2 and cancer vaccines inducing the secretion of INF- γ from T-cells (Garbe et al., 2011)

Apoptosis and cancer 

Programmed cell death biological process inherent cellular programme that leads the cell to self destruct.



Key modulator to tissue homeostasis balancing cell division

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Triggered by stress signals - proinflammatory cytokines - toxins - lack of growth factors - viral infection - hypoxia - DNA damage

Apoptosis and cancer Disregulation of apoptotic mechanisms and pathways is a premium cause of carcinogenesis in many cases

understanding cell rescue mechanisms

Designing of novel therapeutic strategies and diagnostic methods

Apoptosis and cancer

(Ashkenazi, 2002)

Cancer treatment 

Surgery



Chemotherapy Cytotoxic meds -DNA replication inhibitors (cyclophosphamide, cisplatin) -Antimetabolites (methotraxate, fluorouracil etc) -Alkaloid factors (cyclophosphamide, cisplatin, dacarbazine) Cytotoxic Antibiotics(doxorubicin, actinomycin D Plant products (paclitaxel, etoposide, irinotecan) Hormones (fosfestrol, megestrol, glycokortikoids, antihestrogens)

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Radiotherapy (DNA damage)

Cancer treatment Immunotherapy 

CTLs transplantation after incubating with IL-2, TGF-β and IL-15 promising results in metastatic melanoma and renal cancer treatment (Rosenberg et al.,2008)



Modifying Dendritic Cells (DC), with cytokines or chemokines induce an effective T- cell recruitment role (Chen et al., 2010)

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Monoclonal antibodies such as anti-EGFR against specific cancer cell receptors

 Immunoapoptins  Immunostimulants

Imiquimod

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(R837) is a (1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine), nucleoside derivative and antiviral compound



Binds to TLR-7 and TLR-8 and acts via the NFkB pathway (Hurwitz and Pincus et al,2003)

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Stimulates both the innate immune system and the adaptive immune system : -Induces nonspecific B-cell proliferation - Increases NK cell activity - Activates macrophages to secrete cytokines - Induces proliferation and differentiation of B lymphocytes

Imiquimod

DNA damage chemotherapeutic drugs

Doxorubicin 

Cytotoxic antibiotic



Topoisomerase II inhibitor



Connected to caspase 9 and apoptosis

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Intermediate dosages are related to p53 dependent pathways.

Etoposide 

Topoisomerase II inhibitor

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Activation of intrinsic apoptosis pathway involving caspase 3 and 9

The inflammasomes NALP3 IPAF

NALP1

AIM2

PYD domain LRR repeats

Procaspase 1

NACHT domain CARD domain

HIN domain

CARDINAL

ASC

The inflammasomes 

Multiprotein complexes forming upon cellular infection or stress

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Promotes the maturation of proinflammatory cytokines, interleukin 1-β and interleukin 18

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NLRP 1, NLRP 3 and IPAF

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AIM 2

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NLRP 3 most widely studied, - activate upon cholesterol crystals, imidazolequinoline compounds, -cytoplasm of B cells, dendritic cells, esophagus, ropharynx, and ectocervix tissues

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NLR family

HIN-200 DNA binding domain

Purpose Stimulating  inflammasome activation via an immunostimulant (imiquimod) 

Apoptosome formation via DNA damage chemotherapeutic drugs (doxorubicin-etoposide)

Tried to identify possible synergistic and/or cooperative effects of the drugs against cancer cell lines and to understand the molecular mechanisms behind this hypothesis.

Methodology

Treatment of cells with various concentrations and combinations of drugs

Observing the phonotypical treatment effect under microscope

Flow cytometry analysis in order to detect early apoptosis

Western blot analysis to further elucidate the apoptotic pathways

Various cancer cell lines studied

Breast cancer

Colon cancer

- MCF 7 - HCC 1143

- HT 29

Hepatocellular carcinoma

Melanoma

- HUH 7

- MeWo

Chemosensitization studies in MCF 7 breast cancer cells untreated

24h

48h

72h

doxorubicin 0,5 μg/ml & R837 5μg/ml

doxorubicin 0,5 μg/ml & R837 10 μg/ml

Chemosensitization studies in MCF 7 breast cancer cells Untreated

24h

48h

72h

etoposide 15 μg/ml

Etoposide 15 μg/ml &Imiquimod 10μg/ml

MCF 7 cells untreated 48 hrs

MCF 7 cells with 1μg/ml doxorubicin treatment and 5μg/ml imiquimod treatment , 48 hrs

percentage of Annexin v positive cells

Chemosensitization studies in MCF 7 breast cancer cells

90 80 70 60 50 40 30 20 10 0

24h 48h 72h Control

R837 10 μg/ml

Doxorubicin R837 5 R837 5 R837 10 R837 10 1μg/ml μg/ml+Doxo μg/ml+Doxo μg/ml+Doxo μg/ml+Doxo 0,5μg/ml 1μg/ml 0,5μg/ml 1 μg/ml

drug concentration

Chemosensitization studies in HCC 1143 breast cancer cells Untreated

24h

48h

72h

Imiquimod 10μg/ml

Doxorubicin 1μg/ml

Chemosensitization studies in HCC 1143 breast cancer cells Etoposide 15 μg/ml

24h

48h

72h

Etoposide 10 μg/ml &Imiquimod 10μg/ml

Doxorubicin 1μg/ml & R837 10μg/ml

percentage of Annexin V positive cells

Chemosensitization studies in HCC 1143 breast cancer cells 70 60 50 40 30

24h

20

48h

10 0 Control

R837 10 Doxorubicin R837 10 μg/ml 1μg/ml μg/ml+ Doxo 1 μg/ml

R837 5 μg/ml+ Doxo 1μg/ml

drug concentration

R837 10 μg/ml+ Doxo 0,5μg/ml

R837 5 μg/ml+ Doxo 0,5μg/ml

MCF 7 cells are sensitive to chemotherapy, and go through apoptosis via cooperation between imiquimod and doxorubicin/ etoposide HCC 1143 cells are more chemoresistant. Cooperation observed between combination treatment?

Chemosensitization studies in HUH 7 hepatocellular carcinoma cells Untreated

24h

48h

72h

Imiquimod 10μg.ml Doxorubicin 1μg/ml

Doxorubicin 1μg/ml &Imiquimod10μg/ml

HUH 7 untreated cells 48hrs

HUH 7 doxorubicin 1μg/ml treated cells, 48hrs of treatment

HUH 7 cells treated with 10 μg/ml Imiquimod, 48 hrs

HUH 7 cells treated with 5μg/ml Imiquimod & 1μg/ml Doxorubicin, 48 hrs

percentage of Annexin V positive cells

Chemosensitization studies in HUH 7 hepatocellular carcinoma cells 90 80 70 60 50 40 30 20 10 0

24h 48h 72h Control

R837 10 Doxorubicin μg/ml 1μg/ml

R837 5 μg/ml+ Doxo 0,5μg/ml

R837 5 μg/ml+ Doxo 1μg/ml

drug concentration

R837 10 μg/ml+ Doxo 0,5μg/ml

R837 10 μg/ml+ Doxo 1 μg/ml

There is apoptosis inducing cooperative action with doxorubicin and imiquimod combination treatment of HUH 7 cancer cells

Chemosensitization studies in MeWo melanoma cells Untreated

24h

48h

72h

etoposide 15 μg/ml

Etoposide 15 μg/ml +Imiquimod 10μg/ml

Chemosensitization studies in MeWo melanoma cells Untreated

24h

48h

72h

Imiquimod 10μg/ml

Doxorubicin 1μg/ml

Chemosensitization studies in MeWo melanoma cells Doxorubicin 1μg/ml & Imiquimod 10μg/ml 24h

48h

72h

Doxorubicin 0,5μg/ml & Imiquimod 5μg/ml

percentage of AnnexinV positive cells

Chemosensitization studies in MeWo melanoma cells 100 90 80 70 60 50 40

24h

30 20

48h 72h

10 0 Control

R837 10 μg/ml

Doxorubicin 1μg/ml

R837 5 μg/ml+ Doxo 0,5μg/ml

R837 5 μg/ml+ Doxo 1μg/ml

drug concentration

R837 10 μg/ml+ Doxo 0,5μg/ml

R837 10 μg/ml+ Doxo 1 μg/ml

Chemosensitization studies in MeWo melanoma cells

percentage of Annexin V positive cells

80 70 60 50 40 24h

30

48h 20 10 0 untreated

Etoposide 15 μg/ml

Imiquimod 10 μg/ml

Etoposide 10 μg/ml+ Imiquimod 5 μg/ml

Etoposide 10 μg/ml+ Imiquimod 10 μg/ml

Type of treatment

Etoposide 15 μg/ml+ Imiquimod 5 μg/ml

Etoposide 15 μg/ml+ Imiquimod 10 μg/ml

No obvious cooperative action between imiquimod and doxorubicin, but high cooperation detection in MeWo cells treated with imiquimod and etoposide

Chemosensitization studies in HT 29 colon cancer cells untreated

24h

48h

72h

Doxorubicin 0,5 μg/ml

R837 5 μg/ml

Chemosensitization studies in HT 29 colon cancer cells Doxorubicin 0,5 μg/ml +R837 5 μg/ml 24h

48h

72h

Doxorubicin 0,5 μg/ml +R837 10 μg/ml

Chemosensitization studies in HT 29 colon cancer cells- Western blot results 118 90

1

2

3

4

5

6

7

50

Tubilin 55kD

36

P53 50 kD

27 Bax 20 kD

20

1.Untreated 2.Etoposide 2 μg/ml 3.R837 5 μg/ml 4.R837 10 μg/ml

5.Etoposide 2 μg/ml+R837 5 μg/ml 6. Etoposide 2 μg/ml+R837 10 μg/ml 7. Etoposide 10 μg/ml+R837 5 μg/ml

HT 29 cells seem to be apoptosis sensitive in combination treatment with imiquimod and doxorubicin, mechanistic apoptotic details need further investigation

Summarizing.. 

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MCF 7 breast cancer cells respond positively to combination chemotherapy of imiquimod and doxorubicin/etoposide HCC 1143 breast cancer cells are resistant to chemotherapy first evidence shows no cooperation

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HUH 7 hepatocellular carcinoma cells go through apoptosis dramatically when imiquimod and doxorubicin is combined

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MeWo cells hypersensitive to chemotherapy, cooperation between imiquimod/doxorubicine cannot be concluded but high apoptosis induction with imiquimod etoposide treatment

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HT 29 cells phenotypically sensitive to cooperation treatment with imiquimod/doxorubicin

Future experiments 

Elucidation of the apoptotic pathways and the molecules recruited during cooperation through Western blot analysis

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RT quantitive PCR further information on the mechanistic apoptotic details and the involvement of inflammasomes.

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Silencing specific pathway targets (caspase 1, caspase 9), investigation of treatment effect

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Further investigation of early apoptosis detection in targeted chemotherapeutic combinations

Many thanks to…

Nesrin Özören, Ph.D, Group Leader Şahru Yüksel Ph.D. Serkan Uğurlu M.D Elif Eren M.Sc. Ulaş Özkurede B.Sc. Yetiş Gültekin B.Sc. Duygu Demiroz B.Sc. Ali Can Sahilioğlu B.Sc

Thank you for your patience Questions?