List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
Larsson DE, Lovborg H, Rickardson L, Larsson R, Oberg K, Granberg D.
Identification and evaluation of potential anti-cancer drugs on human neuroendocrine tumor cell lines. Anticancer Res 2006; 26(6B):4125-9.
II Larsson DE, Hassan S, Larsson R, Oberg K, Granberg D.
Combination analyses of anti-cancer drugs on human neuroendocrine
tumor cell lines. Cancer Chemother Pharmacol. 2009 Dec;65(1):5-12.
III Larsson DE, Wickström M, Hassan S, Oberg K, Granberg D.
The cytotoxic agents NSC-95397, brefeldin A, bortezomib and sanguinarine induce apoptosis in neuroendocrine tumors in vitro.
Anticancer Res. 2010 Jan; 30(1):149-56.
IV Larsson DE, Hassan S, Oberg K, Granberg D.
The Cytotoxic Effect of Emetine and CGP-74514A studied with the Hollow Fiber Model and Array Scan Assay in Neuroendocrine Tumors In
Vitro.
Submitted
Reprints were made with permission from the respective publishers.
Additional Papers
I.
Aronsson DE, Muhr C. Quantification of sensitivity of endothelial
cell markers for the astrocytoma and oligodendroglioma tumours.
Anticancer Res. 2002 Jan-Feb; 22(1A): 343-346.
II.
Larsson DE, Gustavsson S, Hultborn R, Nygren J, Delle U, Elmroth
K. Chromosomal damage in two X-ray irradiated cell lines: influence of cell cycle stage and irradiation temperature. Anticancer Res.
2007 Mar-Apr;27(2):749-753.
Contents
Introduction ..................................................................................................... 9
Classification of Neuroendocrine Tumors ................................................. 9
Cell death processes ................................................................................. 11
Basic aspect of the cancer drugs investigated .......................................... 11
Brefeldin A .......................................................................................... 12
Emetine ................................................................................................ 12
Bortezomib .......................................................................................... 13
Sanguinarine chloride .......................................................................... 13
NSC 95397 .......................................................................................... 13
CGP-74514A hydrochloride ................................................................ 14
Measurement of Cytotoxicity ................................................................... 14
Multiparametric evaluation of apoptosis .................................................. 15
Cytotoxicity Measurement by Hollow Fiber assay .................................. 16
Aims of the thesis.......................................................................................... 17
Material and Methods ................................................................................... 18
Human tumor cell lines (paper I, II, III and IV) ....................................... 18
Drugs and Reagents (paper I, II, III and IV) ............................................ 18
FMCA (fluorometric microculture cytototoxicity assay) (paper I, II) ..... 20
IC50-values (paper I, II) ....................................................................... 20
Combination studies (paper II) ............................................................ 21
Array Scan (multiparametric apoptosis assay) (paper III, IV) ................. 22
Hollow Fiber Assay (paper IV) ................................................................ 23
Statistical analysis (paper I, II and IV) ..................................................... 23
Results and Discussion ................................................................................. 24
The Screening study (paper I) .................................................................. 24
The combination analyses (paper II) ........................................................ 25
NSC-95397 .......................................................................................... 26
Emetine ................................................................................................ 27
CGP-74514A hydrochloride ................................................................ 29
Brefeldin A .......................................................................................... 29
Sanguinarine chloride .......................................................................... 29
Array Scan (multiparametric apoptosis assay) (paper III, IV) ................. 31
Hollow Fiber Assay (paper IV) ................................................................ 31
Future perspectives ....................................................................................... 32
Summary of the Thesis in Swedish ............................................................... 34
Populärvetenskaplig sammanfattning på svenska .................................... 34
Detaljer om ingående delarbeten .............................................................. 34
Delarbete 1 ........................................................................................... 35
Delarbete 2 ........................................................................................... 35
Delarbete 3 ........................................................................................... 36
Delarbete 4 ........................................................................................... 36
Acknowledgements ....................................................................................... 38
References ..................................................................................................... 41
Abbreviations
BON-1
CI
DMEM
DMSO
FCS
FDA
FMCA
hTERT-RPE1
hTERT
NCI-H727
NCI-H720
IC50
MMP
PBS
SEM
SI
A human pancreatic carcinoid cell line
Combination Index
Dulbecco’s Modification of Eagle’s Medium
Dimethyl sulfoxide
Fetal Calf Serum
Fluoroscein Diacetate
Flurometric Microculture Cytotoxicity Assay
Normal human retinal pigment epithelial cell line
Human telomerase reverse transcriptase
A human typical bronchial carcinoid cell line
A human atypical bronchial carcinoid cell line
Inhibitory Concentration 50% (resulting in 50% survival)
Mitochondrial Membrane Potential
Phosphate buffered saline
Standard Error of the Mean
Survival Index
Introduction
Cancer is characterized by uncontrolled division of cells and the ability of
these cells to invade other tissues, either by direct growth into adjacent tissue
through invasion or by implantation into distant sites by metastasis (1). Over
the last decades, intense research has been done to identify the molecular and
genetic changes of cancer cells, and the pathogenesis of neoplasia. This has
lead to identification of oncogenes, tumor suppressor genes and other molecular mechanisms explaining how the cancer cells growe survive and proliferate (2).
Classification of Neuroendocrine Tumors
Neuroendocrine tumors (NETs) derive from the neuroendocrine cell system,
which is widely distributed in the body and form a heterogeneous group of
malignancies, which differ from each other in their biology, prognosis and
histopathological differences (3-5). The broad heterogeneity characterizing
NETs has always posed problems regarding their correct classification. Neuroendocrine tumors (NETs) were earlier characterized by the production of
peptide hormones that could give different endocrine symptoms. NETs were
classified as functioning or non-functioning. Neuroendocrine tumors (NETs)
have classically been divided into carcinoids and endocrine pancreatic tumors. Carcinoids were further subdivided, depending on the localization of
the primary tumor, into: foregut carcinoids originating from the lungs, thymus, stomach and duodenum (in addition, endocrine pancreatic tumors belong to the foregut group); midgut carcinoids localized in the appendix, jejunum, ileum and proximal colon; and hindgut carcinoids, which originate
from the distal colon and rectum. The most frequent sites of NETs are the
gastrointestinal tract (70%) and the bronco pulmonary system (25%) (6).
Recently, a new classification of NETs has been proposed and adopted by
the World Health Organization (WHO) (7). In order to explain the natural
history of NETs more adequately, this classification is based on a series of
histopathological and biological characteristics: cellular grading, primary
tumor size and site, cell proliferation markers, local or vascular invasivity,
and the production of biologically active substances. The World Health Organization (WHO) classification scheme places neuroendocrine tumors into
three main categories: Well differentiated neuroendocrine tumors, further
9
subdivided into tumors with benign and those with uncertain behavior characterized by a low grade of malignancy, well differentiated (low grade)
neuroendocrine carcinomas with low-grade malignant behavior which are
more aggressive because of the presence of metastases and poorly differentiated neuroendocrine carcinomas with a high grade of malignancy and a
poor prognosis.
This classification enables neuroendocrine tumors in the gastroenteropancreatic tract to be diagnosed easily, but unfortunately it is not applicable to
lung tumors (8, 9).
The classification of lung tumors is currently based on that of Travis et al.
(10) who recognized the following categories: typical carcinoid, atypical
carcinoid, small-cell lung cancer (SCLC) and large-cell neuroendocrine carcinoma (LCNC). Typical bronchial carcinoids are more benign than atypical
carcinoids, but both types are able to metastasize to regional lymph nodes or
distantly to the liver, bones or brain (11, 12). SCLC and LCNC are poorly
differentiated neuroendocrine carcinomas with a high frequency of metastasis and poor prognosis. Carcinoids are tumors able to produce hormones and
they mainly occur in the gastrointestinal tract, a substantial percentage however is found in the bronchopulmonary system (13). Pancreatic endocrine
tumors, also known as islet cell carcinomas, are another type of neuroendocrine tumor which represents 1-2% of the pancreatic neoplasm’s (14). Pancreatic endocrine tumors may cause endocrine syndromes from excessive
production of hormones, such as insulin, gastrin, glucagon or vasoactive
intestinal polypeptide (VIP). Although localized carcinoids or islet cell tumors are surgically manageable, metastatic disease is present in nearly 50%
of patients at the time of diagnosis and cause significant mortality (15).
Experiences of patients with metastatic bronchial carcinoids and pancreatic endocrine tumors have given indication of poor response rates on available treatments (16). Medical treatment of patients with metastatic endocrine
pancreatic tumors includes different chemotherapy combinations and biotherapy such as alpha-interferon and somatostatin analogs. Streptozocin combined with doxorubicin or 5-flourouracil has generated partial remissions in
40-60% of the patients giving a median survival of two years in patients with
advanced disease. Cisplatin plus etoposide have also demonstrated significant antitumor effect in patients with endocrine pancreatic tumors. Alpha
interferon causes significant tumor reduction in about 15% of patients with
long duration, up to three years. Octreotide rarely leads to objective responses (17). The use of the various chemotherapeutic agents, such as doxorubicin, 5-fluorouracil, cisplatin, carboplatin, etoposide streptozotocin and
temozolomide has led to minimal responses in the treatment of patients with
lung carcinoids (18,19) and pancreatic endocrine tumors (20,21), mostly of
short duration. Even if an initial response is obtained in patients with malignant endocrine pancreatic tumors as well as bronchial carcinoids treated with
chemotherapy or biotherapy, resistance to treatment sooner or later occurs.
10
There is thus a need for better treatments in patients with malignant neuroendocrine tumors.
Cell death processes
One target in the research field of oncology is to identify molecules important for apoptosis regulation in tumor cells (22). In order to identify and evaluate promising new anticancer drugs, it is important to rapidly and easily
identify substances with apoptosis-inducing properties. Apoptosis, programmed cell death, is the necessary mechanism complementary to proliferation that ensures homeostasis of all tissues. In recent years, the molecular
machinery responsible for apoptosis has been elucidated, revealing a family
of intracellular proteases, one of the most important groups of proteins involved in apoptosis, the cystein aspartate-specific proteases also called caspases, which are responsible directly or indirectly for the morphologic and
biochemical changes that characterize the phenomenon of apoptosis (23,24).
Caspases are activated by different toxic stimuli. Three major pathways have
been elucidated so far, which all result in the activation of caspase-3. One is
the mitochondrial/cytochrome C pathway, largely mediated through Bcl-2
family members, which results in activation of Apaf-1, caspase-9, and then
caspase-3. The second pathway involves ligation of members of the TNFreceptor family (e.g., Fas, TRAIL receptors) and activates caspase-8 and
subsequently caspase-3. Finally, granzyme B (a cytolytic T-cell product)
directly cleaves and activates several caspases, resulting in apoptosis (25,
26). Regardless of the pathways, it ends with DNA fragmentation with formation of apoptotic bodies, which are phagocysed by macrophages (27).
Basic aspect of the cancer drugs investigated
Cancer chemotherapy is the use of chemical compounds with various mechanism of action against growth and survival of tumor cells. It is used to
cure or relieve tumor-related symptoms and prolong the life. Chemotherapy
can be given alone as the only treatment or in combination with surgery and
radiotherapy (28). In this thesis eleven anticancer drugs have been evaluated
to have effect on two bronchial carcinoid cell lines and a pancreatic carcinoid cell line in vitro. Six of these drugs have been further investigated.
Figure 1 shows the molecular structures of the drugs investigated in this
thesis.
11
________________________________________________________
Brefeldin A
Emetine
Bortezomib
NSC 95397
CGP-74514A hydrochloride
Sanguinarine chloride
________________________________________________________
Figure 1. Molecular structures of Brefeldin A, Bortezomib, CGP-74514A, Emetine,
NSC 95397 and Sanguinarine
Brefeldin A
Brefeldin A was initially introduced as an antiviral antibiotic (29) which is
currently known to play a regulatory role in the intracellular transport system. Its biological activities have been reported to inhibit intracellular protein transport from the endoplasmic reticulum (ER) to the cis-Golgi apparatus (30), to promote reversible disassembly of the Golgi complex (31) and to
facilitate redistribution of Golgi-associated proteins to the ER (32). A possible antitumor effect of Brefeldin A has also been proposed (33).
Emetine
Emetine is a potent protein synthesis inhibitor and is clinically used in the
treatment of protozoan infection. Emetine has shown promise as an antitumor agent without bone marrow suppression (34-36).
The cytotoxicity of emetine is due to inhibition of protein biosynthesis in
eukaryotic ribosomes (37) and interaction with DNA (38) Evidence of emetine's activity against tumor cells first came to light in 1918 (39) and its use
in phase I and II clinical cancer trials began over 30 years ago (40- 42).There
are some publications suggesting induction of apoptosis by emetine in the
12
human leukemic cell line (U937) (43), human lung epithelial A549-S cells
(44) and rat hepatocytes (45). Earlier studies have also shown that emetine is
a strong inducer of apoptosis and caspase activity, comparable to the cytotoxic effect of cisplatin. Moreover, emetine enhanced the cytotoxic effect of
cisplatin and increased cisplatin-induced apoptosis in leukemia cells (42).
Thus, emetine could be a suitable cytotoxic agent in cancer therapy, e.g. to
overcome multidrug resistance or to take advantage of synergistic effects in
order to minimize side-effects due to the high dosage of other cytotoxic
agents.
Bortezomib
Bortezomib (Velcade®), a proteasome inhibitor, has shown activity in early
clinical trials among patients with Non- Hodgkin’s lymphoma and multiple
myeloma.
In a phase II trial, 50% of patients with recurrent myeloma who received
1.3 mg/m2 Bortezomib responded with complete inhibition of myeloma cell
growth (46).
In vitro and in vivo (murine xenograft) studies have shown that bortezomib is active against a variety of malignancies, including hematologic malignancies and solid tumors (ie, breast, prostate, lung, pancreas, colon, ovarian, and head and neck cancers). Bortezomib has demonstrated activity as a
single agent and in combination with several cytotoxic agents, such as 5fluorouracil, doxorubicin, and docetaxel, and with radiation, enhancing both
chemotherapy- and radiation-therapy-induced apoptosis. Bortezomib has
also shown activity in some cell lines resistant to standard therapies (47-57).
Sanguinarine chloride
Sanguinarine chloride, a Na+/K+-ATPase inhibitor has been shown to possess antimicrobial, antioxidant, anti-inflammatory, and antitumor properties
and is widely used in toothpaste and mouthwash to prevent/treat gingivitis
and other inflammatory conditions of the mouth (58-60). Sanguinarine was
also identified as a potential inhibitor of survivin that selectively kills prostate cancer cells over “normal” prostate epithelial cells. Survivin is selectively over expressed in most common human cancers and is a member of the
inhibitor of apoptosis protein family. Studies have shown that sanguinarine
induces apoptosis in various human cancer cells, including prostate cancer
(61-64).
NSC 95397
NSC 95397 is described as a Cdc25 phosphatase inhibitor (65). Progression
through the cell division cycle and transitions between its various phases are
13
regulated by the activation of specific cyclin-dependent kinase (CDK) complexes by CDC25 phosphatases (66).
As Cdc25 phosphatases promote cell cycle progression and are over expressed in numerous rapidly dividing cancer cells, one might expect a correlation between over expression and the rate of proliferation. In fact, no correlation has been seen the majority (67-72).Thus, the role of Cdc25 over expression is more complicated than that of a simple driver of cell proliferation. It is quite likely that Cdc25 over expression in tumors is required to
circumvent many of the checkpoints that would otherwise hinder cell proliferation.
NSC95397 is the most potent Cdc25 inhibitor to date (IC-50s of 22–125
nM). This compound blocks the G2/M transition and shows growth inhibition against human carcinoma cell lines (66).
CGP-74514A hydrochloride
Cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) work
simultaneously to regulate the cell cycle. CGP74514A is a new trisubstituted
purine derivative that has been reported to function as a potent and selective
inhibitor of CDK1 (73). For progression of the cell cycle phases, formation
of complexes between cyclins and CDKs is necessary and CKIs have an
inhibitory role against this. In addition to inhibiting cell cycle progression,
CDK inhibitors are effective inducers of apoptosis in neoplastic cells (74).
Measurement of Cytotoxicity
The cytotoxic effect of different drugs can be studied by the fluorometric
microculture cytototoxicity assay (FMCA) (Figure 2). The assay is based on
the presence of esterases in viable cells that convert colourless fluorescein
diacetate (FDA) to fluorescent fluorescein. Fluorescence intensity after drug
exposure thus indicates the amount of surviving cells when compared to
unexposed control cells.
14
Figure 2. Schematic illustration of the main steps of the Fluorometric Microculture
Cytotoxicity Assay (FMCA)
Multiparametric evaluation of apoptosis
The current focus of drug discovery in the area of oncology is to identify
molecules important for apoptosis regulation in tumor cells (22). Study of
mechanistic action for tumor cell death; how the cells go to apoptosis or
necrosis can be done by in vitro study. Thus, in the process of identifying
and evaluating promising new anticancer agents, it is important to rapidly
and easily identify substances with apoptosis-inducing properties. One approach to identify and evaluate substances that induce apoptosis would be to
use automated information-rich image-based analysis, Array Scan, multiparametric apoptosis assay often called high-content screening (75). This assay
is based on measurement of fluorescence intensity and localization on a cellby-cell basis.
15
Cytotoxicity Measurement by Hollow Fiber assay
Many of the in vitro models used in preclinical anticancer drug development
are based on monolayer or suspension cultures of human tumor cell lines.
These models are technically simple and useful in selecting potentially active compounds for further study. However, they do not mimic the complex
microenvironment, heterogeneity and proliferative properties of solid tumors
and this may contribute to incorrect predictions of in vivo efficacy (76). Anticancer drugs can be effective in solid tumors only if they can penetrate
several cell layers and retain their activity in the tumor microenvironment
(77).To address this, three-dimensional in vitro solid tumor models have
been developed such as the in vitro Hollow Fiber models (78). This assay,
which is based on culturing tumor cells in polyvinylidine fluoride hollow
fibers, was developed by Hollingshead (79).
The introduction of hollow fibers represents an important advancement in
the field of in vitro cytotoxicity. It is believed that in vitro testing methods
are useful, time and cost effective tools for drug discovery but many of the
available tests are not effective for examining both time and concentration,
and do not closely mimic human kinetics. This is because they do not properly take into account pharmacodynamic actions (what a drug does to the
body) and pharmacokinetic actions (what a body does to the drug). The Hollow Fiber model has changed this. The hollow fiber model has exceeded the
standard in vitro cytotoxity methods by mimicking changes in the concentration of the drug over time as they occur in humans. Hollow Fiber technology
offers higher reproducible control of both concentration and time of drug
exposure in complex growth, infection, treatment and sampling regimens.
This system permits more realistic simulation of in vivo drug effects in a
dynamically controlled system providing data that more accurately reflects
biological responses (80).
16
Aims of the thesis
The overall aims of the thesis were to preclinically investigate new anticancer drugs on neuroendocrine tumors, based on mechanism of action, apoptosis
and the cytotoxic effect on the cell lines using single drug or combination
treatment. The overwhelming aim is to find more effective treatment for
patients with endocrine tumors.
The specific aims were to:
1.
Examine new anti-cancer drugs with effect on bronchial carcinoids and
pancreatic endocrine tumors.
2.
Evaluate potential synergistic effects for the new anti-cancer drugs,;
NSC 95397, emetine, CGP-74514A hydrochloride, brefeldin A and
sanguinarine chloride, when combined with four standard cytotoxic
drugs already used in the clinic for treatment of neuroendocrine tumors.
3.
Study the mechanistic effect for the anti-cancer drugs,; NSC 95397,
bortezomib, brefeldin A and sanguinarine chloride for the apoptosis and
morfologic changes with the ArrayScan assay.
4.
Characterize in vitro the cytotoxic activity of emetine and CGP-74514A
with Hollow Fiber assay and the apoptosis and morphologic changes
with the ArrayScan assay.
17
Material and Methods
Human tumor cell lines (paper I, II, III and IV)
The human pancreatic carcinoid cell line BON-1 cells, derived from a lymph
node metastasis of a human pancreatic carcinoid tumor, was cultured in a
(1:1) nutrient mixture of Dulbecco’s Modification of Eagle’s Medium
(DMEM) and Kaighn’s modification medium (F12K) (Invitrogene, Sweden).
The human typical bronchial carcinoid cell line NCI-H727 and the human
atypical bronchial carcinoid cell line NCI-H720 was obtained from ATCC
(LGC Promochem, Sweden) and maintained in RPMI 1640 medium (Invitrogene, Sweden). The cell lines were supplemented with 10% heatinactivated Fetal Calf Serum (FCS), 1% glutamine and 1% penicillin/streptomycin (Sigma Aldrich) and cultured in a 5% CO2 humidified atmosphere at 37 ºC. For comparison, we also studied a normal human retinal
pigment epithelial cell line, hTERT-RPE1 (a human RPE cell line that stably
expresses human telomerase reverse transcriptase (hTERT)) in study I. The
epithelial cell line hTERT-RPE1 was cultured in DMEM.
Drugs and Reagents (paper I, II, III and IV)
In paper I; as a screening study, we first evaluated 1280 drugs obtained from
the LOPAC1280TM library (Sigma Aldrich, St. Louis, MO). The library was
screened at 10 µM in three tumor cell lines. Drugs with survival index (SI)
above 60% were eliminated from further studies. Survival index was defined
as the fluorescence of experimental wells in percent of control wells with
blank values subtracted. SI = 100 x (treated cells - blank)/ (control cells –
blank). The primary screening resulted in 18 candidate drugs with SI-value
of less than 60%. These drugs were chosen for further dose-response experiments in the three tumor cell lines. The drugs selected from the initial
screening, their mechanisms of action and the solvents are shown in (Table
1) (paper I). Drugs were purchased from Sigma-Aldrich. Doxorubicin was
supplied by the local pharmacy (Uppsala, Sweden). All drugs were tested at
five 10-fold dilutions ranging from 0.01 to 100 µM or 0.001 to 10 µM for
the tumor cell lines and the epithelial cell line hTERT-RPE1.
18
Table 1. The tested drugs, mechanisms of action and solvents.
Drug
Solvent
Mechanism of action
Apomorphine
Bay 11-7085
Sterile water
DMSO
Bortezomib
Brefeldin A
DMSO
Ethanol, 95%
Camptothecin
Cantharidin
CGP- 74514A
Colchicine
Doxorubicin
Emetine
Idarubicin
-Lapachone
DMSO
DMSO
DMSO
Sterile water
PBS
Sterile water
Sterile water
DMSO
Mitoxantrone
Ethanol, 95%
NSC 95397
DMSO
Ouabain
Parthenolide
Sanguinarine chloride
Vincristin
Sterile water
DMSO
Methanol
Non-selective dopamine receptoragonist
Inhibits cytokine induced IkB· (inhibitor of NF k B)
phosphorylation
Proteasome inhibitor-ubiquitin-proteasome pathway
Inhibitor of protein translocation from the endoplasmic
reticulum (ER) to the Golgi apparatus
DNA topoisomerase I inhibitor
Protein phosphatase 2A inhibitor
Cyclin-dependent kinase-1 (Cdk1) inhibitor
Inhibitor of tubulin (prevents tubulin polymerization)
Inhibitor of DNA topoisomerase II
Protein synthesis inhibitor at the level of translation
Inhibitor of DNA metabolism
Inhibition or activation of DNA topoisomerase and
inhibition of NF-kB activity
Inhibitor of DNA metabolism, DNA synthesis inhibitor
Selective, irreversible Cdc25 dual specificity phosphatase inhibitor
Inhibitor of NA+/K+-ATPase
Inhibits serotonin release from platelets
Inhibitor of Na+/K+-ATPase
PBS
Inhibitor of tubulin (inhibit microtubule assembly)
DMSO, dimethyl-sulphoxide; PBS, phosphate-buffered saline, pH 7.4.
In paper II; five of these compounds, with different mechanisms of action,
were chosen for further studies aiming at evaluating potential synergistic
effects when combined with four standard cytotoxic drugs already used in
the clinic for treatment of neuroendocrine tumors. The five chosen compounds were NSC-95397, a selective Cdc25 dual specificity phosphatase
inhibitor; emetine, a protein synthesis inhibitor and DNA interacting agent;
CGP- 74514A hydrochloride, a cyclin-dependent kinase-1 inhibitor; brefeldin A, the inhibitor of the protein transport from the endoplasmic reticulum
to the Golgi apparatus and sanguinarine chloride, a Na+/K+-ATPase inhibitor (59,38). The four standard drugs were doxorubicin and etoposide which
are DNA topoisomerase II inhibitors, oxaliplatin which is alkylating and
inhibits DNA synthesis by disrupting DNA replication and transcription, and
docetaxel, a microtubule stabilizer and mitotic progression inhibitor. The
studies were performed on three neuroendocrine tumor cell lines; the atypical bronchial carcinoid NCI-H720, the typical bronchial carcinoid NCI-H727
19
and the human pancreatic carcinoid cell line BON-1. The compounds NSC95397, emetine, CGP-74514A hydrochloride, brefeldin A, sanguinarine
chloride and the standard drugs; etoposide, oxaliplatine and docetaxel were
purchased from Sigma-Aldrich, while doxorubicin was supplied by the local
pharmacy (Uppsala, Sweden). All compounds and drugs were prepared according to the manufacturer’s instructions.
In paper III, measurement of apoptosis was used to investigate the apoptosis resulting from NSC 95397, brefeldin A, bortezomib and sanguinarine
in the typical bronchial carcinoid NCI-H727 and the human pancreatic carcinoid cell line BON-1.
In paper IV, measurement of apoptosis was used to investigate the apotosis for emetine and CGP-74514A and an in vitro hollow fiber model were
used to study the effect and mode of induced cell death of emetine and CGP74514A in the human endocrine carcinoids, typical lung carcinoid cell line
and atypical lung carcinoid cell line.
FMCA (fluorometric microculture cytototoxicity assay)
(paper I, II)
The fluorometric microculture cytototoxicity assay (FMCA) is a method to
study the cytotoxic effect or sensitivity of different drugs. Tumor cells are
cultured in drug-prepared 384-well plates. Three columns without drugs
served as controls and one column with medium only served as blank. The
plates were incubated at 37º C for 72 h after which they were analyzing using the FMCA. The assay is based on the presence of esterases in viable cells
that convert colorless fluorescein diacetate (FDA) to fluorescent fluorescein
by cells with intact plasma membranes. The fluorescence, which is proportional to the number of living cells, is measured. The drug effect is expressed
as survival index (SI%) i.e. the fluorescence of drug exposed cultures as a
percentage of an unexposed control with blank values subtracted.
IC50-values (paper I, II)
The IC50 values (inhibitory concentration 50%), estimated from the log concentration–effect curves in Graph Pad Prism (GraphPad software Inc., CA,
USA) using non-linear regression analysis, for all tested drugs in the three
cell lines were determined. The concentration range for the individual drugs,
the mean IC50 values for all 3 cell lines, as well as their drug ratios (test
drug/standard drug) is shown in Table 2 (paper II).
The mean of the estimated IC50 of the drug in the 3 cell lines for each
drug was used to select its concentration range for the combination studies in
study II.
20
Table 2. The tested drugs, concentration range, mean IC50-values for all three neuroendocrine tumor cell lines and drug ratios.
Drug
Concentration range
tested (µM)
IC50-value (µM)
NSC-95397
Doxorubicin
Etoposide
Oxaliplatin
Docetaxel
Emetine
Doxorubicin
Etoposide
Oxaliplatin
Docetaxel
CGP-74514A
Doxorubicin
Etoposide
Oxaliplatin
Docetaxel
BrefeldinA
Doxorubicin
Etoposide
Oxaliplatin
Docetaxel
Sanguinarine
Doxorubicin
Etoposide
Oxaliplatin
Docetaxel
0.34–86
0.19–48
6.6–1,696
2.5–630
0.62–160
0.0062–1.6
0.19–48
6.6–1,696
2.5–630
0.62–160
0.12–32
0.19–48
6.6–1,696
2.5–630
0.62–160
0.0062–1.6
0.19–48
6.6–1,696
2.5–630
0.62–160
0.062–16
0.19–48
6.6–1,696
2.5–630
0.62–160
5.4
3.0
106
39
10
0.1
3.0
106
39
10
2.0
3.0
106
39
10
0.1
3.0
106
39
10
1.0
3.0
106
39
10
Drug ratios (tested
drug/standard drug)
1:0.5
1:19
1:7
1:2
1:30
1:1,06
1:394
1:100
1:2
1:53
1:20
1:5
1:30
1:1,06
1:394
1:100
1:3
1:106
1:39
1:10
Combination studies (paper II)
The combination studies were designed as suggested in the CalcuSyn software manual (81). We investigated nine different concentrations of each
drugs by diluting the highest concentration two-fold to span a wide effect
range, on both side of IC50. With this design, the drugs were combined at
equipotent concentrations with a fix ratio. IC50 of one drug were combined
with IC50 of the other drug and so on. Fixed concentration ratios of the drugs
were used with two-fold serial dilutions in nine steps for combinations and
for single drug containing wells.
To characterize the combination effects between the 5 compounds and the
4 standard cytotoxic agents, data were analyzed according to the medianeffect method of Chou and Talalay, using the software CalcuSyn Version 2
(Biosoft, Cambridge, UK) (82). Each dose–response curve (individual agents
as well as combinations) was fit to a linear model using the median effect
21
equation. The extent of drug interaction between the drugs was expressed
using the combination index (CI) for mutually exclusive drugs:
CI = d1/D1 + d2/D2
where D1 and D2 represent the concentration of drugs 1 and 2 alone, required
to produce a certain effect and d1 and d2 are the concentration of drugs 1 and
2 in combination required to produce the same effect. Different CI values are
obtained when solving the equation for different effect levels and 75% effect
was chosen for presentation. Synergy was defined as CI significantly lower
than 1 and antagonism as CI significantly higher than 1. When the confidence interval included 1 the interaction was defined as additive.
Array Scan (multiparametric apoptosis assay)
(paper III, IV)
Cell death characterististics, can be studied by a multiparametric single-cell
assay. We have studied the mechanistic effect for the cell death; apoptosis
for the most interesting drugs for the to neuroendocrine tumor cell lines
BON-1 and NCI-H727. This was done with the multiparametric apoptosis
assay, Array Scan HCS. Cells are seeded and exposed to drugs in 96-well
plates and probes are added to stain apoptotic markers: FAM-DEVD-FMK
to stain activated caspase-3, MitoTracker Red to evaluate mitochondrial
membrane potential (MMP) and Hoechst 33342 to stain nucleus. Analysis
are performed by the ArrayScan high content screening system (Cellomics
Inc.) which is a computerized automated fluorescence imaging microscope
that automatically identifies stained cells and reports the intensity and distribution of fluorescence in individual cells. Automatic focusing, image acquistition and analysis are performed to collect data on a user-defined number of
cells. Images and data regarding intensity and texture of the fluorescence
within the individual cells, as well as the average fluorescence of the cell
population within a well are stored in a database for easy retrieval and analsysis.
In this work, we present evaluation of caspase-3 activity, fragmentation/condensation and nuclear morphology in cells exposed to the cytotoxic
drugs, NSC 95397, brefeldin A, sanguinarine chloride, emetine and CGP74514A hydrochloride.
22
Hollow Fiber Assay (paper IV)
We have studied if the effect of the drugs on the endocrine carcinoid cell
lines can be transferred to the solid tumors by the Hollow Fiber Assay.
The assay is based on implanting tumor cells cultured in polyvinylidine
fluoride hollow fibers in vitro or in vivo for the living cell density assessment.
In paper IV we have applied an in vitro Hollow Fiber model for the assessment of growth and drug sensitivity for the bronchial carcinoid cell lines.
The effect of the assay can be analyzed by fluorometric microculture cytototoxicity assay (FMCA).
Statistical analysis (paper I, II and IV)
The IC50-values for drugs in the cell lines were determined from log concentration effect (survival index %) curves in GraphPad Prism using non-linear
regression analysis. Statistical analysis was performed using the GraphPad
Prism software. Comparison of activity between two groups, here the activity in the cell lines, was made with an unpaired Student’s t test and ANOVA
with Bonferroni post-hoc test were used to compare three or more treatments
groups. Significance level was set to p < 0.05. Comparison of activity between two groups in paper II was made with two-sided unpaired Student’s t
test. One-sample t tests were used to determine if the CI differed from 1. SI
values at different drug concentrations for the various tumor cells tested
were compared using an unpaired Student's t-test in paper IV.
23
Results and Discussion
The Screening study (paper I)
The aim of the Paper I was to examine new anti-cancer drugs with effect on
bronchial carcinoids and pancreatic endocrine tumors. The fluorometric microculture cytototoxicity assay (FMCA) were used for screening of 1280
drugs from the library (LOPAC1280™) on the three neuroendocrine cell lines
and the normal human retinal pigment epithelial cell line, hTERT-RPE. The
screening study resulted in 18 candidate drugs with SI-value of less than
60%. To assess the activity of the hit compounds, dose-response experiments
were performed. The activity of the compounds was determined by their
IC50-values (the concentration of the drug where > 50% of the cells dies).
Compounds with IC50-values < 10 µM were selected as active. Eleven of 18
compounds were sensitive with IC50-values < 10 µM, shown in Table 3
(study I). Brefeldin A, emetine, bortezomib and idarubicin were the most
active agents in vitro, with IC50 values < 1 µM in all four cell lines, while
sanguinarine chloride showed IC50 values between 0.5 and 2.µM. In addition,
Bay 11-7085, mitoxantrone, doxorubicin, -lapachone, CGP-74514A and
NSC 95397 showed IC50 values < 10 µM in the three tumor cell lines.
To conclude, in Paper I, our results indicated that in vitro screening using
annotated compound libraries may be used for identification of compounds
with antitumor activity in neuroendocrine tumor models. Our study demonstrated that Bay 11-7085, bortezomib, brefeldin A, CGP-74514A, doxorubicin, emetine, idarubicin, -lapachone, mitoxantrone, NSC 95397, and sanguinarine showed antitumor effect in the human bronchial carcinoid and
pancreatic carcinoid cell lines in vitro.
24
Table 3. In vitro sensitivity of the drugs with IC50 values <10µM in the tumor cell
lines
Drug
Brefeldin A
Emetine
Bortezomib
Idarubicin
Sanguinarine
Bay 11-7085
Mitoxantrone
Doxorubicin
-Lapachone
CGP74514A
NSC 95397
NCI-H720
IC50
0.071
0.094
0.55
0.71
0.57
1.6
1.6
1.4
2.1
2.2
1.4
NCI-H727
IC50
0.092
0.15
0.63
0.87
1.0
3.4
3.5
5.4
2.7
3.2
8.3
BON-1
IC50
0.52
0.11
0.38
0.99
1.8
2.2
2.1
3.4
3.0
1.9
8.9
hTERT-RPE1IC50
0.29
0.40
0.73
0.26
1.5
0.97
0.69
2.7
3.2
13
3.5
H720, atypical bronchial carcinoid cell line; H727, typical bronchial carcinoid cell line; BON1, pancreatic carcinoid cell line; hTERT-RPE1, normal human retinal pigment epithelial cell
line.
The combination analyses (paper II)
The aim of Paper II was to evaluate potential synergistic effects for the new
anti-cancer drugs; NSC 95397, brefeldin A and sanguinarine chloride, when
combined with four standard cytotoxic drugs already used in the clinic for
treatment of neuroendocrine tumors. Synergistic effect is the interaction
between two or more substances that produces an effect greater than the sum
of their individual effects. It is opposite of antagonism. Additive effect is the
term used when two or more drugs are taken at the same time and the action
of one plus the action of the other results in an action as if just one drug had
been given. Antagonism is a phenomenon where two or more substances in
combination have an overall effect which is less than the sum of their individual effects.
Table 4 summarizes the combination effect for NSC-95397, emetine,
CGP-74514, brefeldin A and sanguinarine combined with the four standard
drugs in the three neuroendocrine tumor cell lines.
The number of synergistic interactions of the five drugs with the four standard drugs in the typical NCI-H727, the atypical NCI-H720 and the pancreatic neuroendocrine cell line BON-1 was 11/20, 7/20 and 5/20 respectively.
25
Table 4. Combination effect of NSC-95397, emetine, CGP-74514A, brefeldin A,
and sanguinarine with the standard chemotherapeutic drugs in neuroendocrine tumor
cell lines: pancreatic carcinoid tumor (BON-1), typical broncial carcinoid (NCIH727) and atypical broncial carcinoid (NCI-H720) in vitro
Combination
BON-1
Effect
NCI-H727
Effect
NCI-H720
Effect
NSC-95397 + Doxorubicin
NSC-95397 + Etoposide
NSC-95397 + Oxaliplatin
NSC-95397 + Docetaxel
Emetine + Doxorubicin
Emetine + Etoposide
Emetine + Oxaliplatin
Emetine + Docetaxel
CGP-74514A + Doxorubicin
CGP-74514A + Etoposide
CGP-74514A + Oxaliplatin
CGP-74514A + Docetaxel
Brefeldin A + Doxorubicin
Brefeldin A + Etoposide
Brefeldin A + Oxaliplatin
Brefeldin A + Docetaxel
Sanguinarine + Doxorubicin
Sanguinarine + Etoposide
Sanguinarine + Oxaliplatin
Sanguinarine + Docetaxel
Synergistic
Synergistic
Additive
Additive
Additive
Synergistic
Synergistic
Synergistic
Additive
Additive
Additive
Additive
Additive
Additive
Additive
Antagonistic
Additive
Additive
Antagonistic
Additive
Synergistic
Synergistic
Synergistic
Synergistic
Additive
Synergistic
Additive
Synergistic
Synergistic
Additive
Additive
Antagonistic
Synergistic
Synergistic
Additive
Synergistic
Synergistic
Additive
Additive
Additive
Synergistic
Synergistic
Synergistic
Additive
Additive
Synergistic
Synergistic
Synergistic
Additive
Additive
Additive
Additive
Antagonistic
Synergistic
Additive
Additive
Additive
Additive
Antagonistic
Antagonistic
________________________________________________________
NSC-95397
The combination of NSC-95397 and the four standard cytotoxic drugs
showed synergy in the two lung carcinoid cell lines NCI-H727 and NCIH720. The interaction with NSC-95397 and doxorubicin or NSC 95397 and
etoposide in the pancreatic carcinoid cell line BON-1 also showed synergy.
The combination with docetaxel showed additive effect in the atypical carcinoid cell line NCI-H720. It was additive with oxaliplatin and docetaxel carcinoid cell line BON-1. The number of synergistic interactions of NSC95397 with all four standard drugs was 9/12.
All NSC-95397 combinations with doxorubicin and etoposide were synergy which may indicate that these combinations may be the possible candidates for further pre-clinical and clinical studies. It has previously been reported that NSC-95397 showed synergy effect with paclitaxel in breast cancer cell line (22) and in the combination of other Cdc25 inhibitors with paclitaxel have additive effect on colon cancer cells (38). Docetaxel is a drug not
commonly used in patients with carcinoids and there have been no published
26
reports, preclinical or clinical, on docetaxel-containing combinations in the
treatment of patients with neuroendocrine tumors. Our results indicate that
docetaxel as well as oxaliplatin are suitable candidates for combination with
NSC-95397 with a synergy and additive effect. It is important to remember
that additive interactions, not only synergistic, could be of clinical benefit if
the drugs have non-overlapping toxicity profiles. For NSC-95397 no reports
are available regarding its toxic side effects. Combining drugs with synergistic or additive interaction effects and non-overlapping side effects may enable dose reduction of toxic chemotherapeutic agents, yet maintaining enough
antitumor effect.
Emetine
The interactions for emetine were mainly synergy with etoposide, oxaliplatin
and docetaxel while they were additive with doxorubicin in all cell lines.
Such combinations are thus interesting objects for further studies. The number of synergistic interactions of emetine with all four standard drugs was
8/12. In Figure 3, emetine and etoposide combinations showing synergism
are visualized as concentration-effect curves, bar graph of chosen concentration, isobolograms and CI plots, in BON-1 (Figure 3 A-D, left panel) and
H727 (Figure 3 E-H, right panel) cell lines.
27
____________________________________________________________
E
120
120
100
100
S u r v iv a l In d e x (% )
S u r vival In d ex (% )
A
80
60
40
Emetine
Etoposide
Combination
20
0
0.001
0.01
0.1
1
80
60
40
Emetine
Etoposide
Combination
20
0
0.001
10
0.01
B
1
10
F
125
100
100
Survival Index (%)
Survival Index (%)
0.1
Concentration Emetine (µM)
Concentration Emetine (µM)
75
50
25
0
Emetine
0.03 M
Etoposide
26 M
75
50
25
0
Combination
C
Emetine
0.05 M
Etoposide
53 M
Combination
G
Isobologram
Isobologram
1.2
0.60
0.40
Emetine
Emetine
0.9
0.6
0.3
0.20
0
0
0
200
ED50
400
600
Etoposide
ED75
800
0
ED90
100
ED50
D
300
ED75
400
500
ED90
H
Emetine + Etoposide - Algebraic estimate
Emetine + Etoposide - Algebraic estimate
1.5
CI +/- 1.96 s.d.
3.0
CI +/- 1.96 s.d.
200
Etoposide
2.0
1.0
1.0
0.5
0
0
0
0.2
0.4
0.6
Fractional Effect
0.8
1.0
0
0.2
0.4
0.6
Fractional Effect
0.8
1.0
_____________________________________________________________
Figure 3.Combination of emetine and etoposide in the human pancreatic carcinoid
cell line, BON-1(A–D, left panel) and in the human typical bronchialcarcinoid cell
line NCI-H727 (E–H, right panel).
28
CGP-74514A hydrochloride
Almost all interactions for CGP-74514A hydrochloride and the standard
cytotoxic drugs were additive. This agent may thus also be a possible
candidate for further studies.
The synergistic interactions of CGP-74514A hydrochloride, with the four
standard drugs were few, 1/12, and the number of additive interactions were
10/12.
Brefeldin A
The most interactions for Brefeldin A were also additive and the synergistic
interactions with the four standard drugs were few, 4/12 while the numbers
of additive interactions were, 6/12.
Sanguinarine chloride
The synergistic interactions of sanguinarine chloride with the four standard
drugs were also few, 1/12, and the numbers of additive interactions were,
8/12 for the three cell lines. The interactions with etoposide were additive,
while the interactions with oxaliplatine and docetaxel showed antagonistic
effect for the atypical carcinoid cell line NCI-H720. Sanguinarine and oxaliplatin combinations showing antagonism are visualized as concentration–
effect curves, bar graph of chosen concentration, isobolograms and CI plots
in Figure 4, in BON-1 (Figure 4 A-D , left panel) and H720 (Figure 4 E-H ,
right panel) cell lines.
To summarize, in paper II, the interaction-effects between the five investigated drugs and the four standard cytotoxic agents were mostly synergistic
or additive. NSC-95397, the Cdc25 dual specificity phosphatase inhibitor
and emetine, the protein synthesis inhibitor and DNA interacting agent had
the most attractive interactions (synergy) in combination with the four standard cytotoxic drugs in the three cell lines tested. The synergy interactions of
NSC-95397 and emetine in the pancreatic and atypical bronchial carcinoid
cell lines is worth noting as the pancreatic carcinoid cell line previously has
been shown to be the least sensitive cell line to the five drugs (59) and clinical studies have demonstrated a worse prognosis for patients with atypical
compared to patients with typical bronchial carcinoids (83).
The three remaining compounds CGP-74514A, brefeldin A and sanguinarine displayed less synergy interactions, with combination indicex above 1 in
most of the interactions with the standard drugs. They thus seem less attractive to combine with the standard chemotherapeutic agents we tested.
29
________________________________________________________
A
E
120
100
80
60
40
Sanguinarine
Oxaliplatin
Combination
20
0
0.01
0.1
1
10
Survival Index (% )
S u r vival In d ex (% )
120
100
80
60
40
Sanguinarine
Oxaliplatin
Combination
20
0
0.01
100
Concentration Sanguinarine (µM)
0.1
1
10
100
Concentration Sanguinarine (µM)
F
B
125
Survival Index (%)
Survival Index (%)
125
100
75
50
25
100
75
50
25
0
Sanguinarine Oxaliplatin Combination
0.5 M
20 M
0
Sanguinarine Oxaliplatin Combination
79 M
2 M
C
G
Isobologram
Isobologram
8.0
10.0
6.0
Sanguinarine
Sanguinarine
8.0
6.0
4.0
4.0
2.0
2.0
0
0
0
200
ED50
400
Oxaliplatin
ED75
0
600
100
ED50
ED90
D
300
ED75
400
500
ED90
H
Sanguarine + Oxaliplatin - Algebraic estimate
Sanguarine + Oxaliplatin - Algebraic estimate
15
4.0
3.0
C I +/- 1.9 6 s.d .
CI +/- 1.96 s.d.
200
Oxaliplatin
2.0
1.0
10
5
0
0
0
0.2
0.4
0.6
Fractional Effect
0.8
1.0
0
0.2
0.4
0.6
Fractional Effect
0.8
1.0
________________________________________________________
Figure 4.Combination of sanguinarine and oxaliplatin in the human pancreatic carcinoid cell line, BON-1 (A–D, left panel) and in the human atypicabronchial carcinoid
cell line NCI-H720 (E–H, right panel).
30
Array Scan (multiparametric apoptosis assay)
(paper III, IV)
Our results show that NSC 95397 bortezomib, brefeldin A, sanguinarine,
emetine and CGP-74514A, induced high level of caspase-3 activity, apoptotic changes in nuclear morphology with nuclear fragmentation/condensation
and a dose-dependent increase in fragmentation/condensation after 24h, 48h
and 72 h in the neuroendocrine cell lines. Cells exposed for drugs for 48 h
showed marked increase in the typical signs of apoptosis: chromatin condensation and nuclear fragmentation with less necrosis. After 24 h, a modest
decrease in MMP was observed for the drugs at the lowest tested concentrations compared to the untreated controls. In summary, in Paper III and IV
our data indicated induction of apoptosis with the tested drugs by intrinsic or
extrinsic pathways in the neuroendocrine tumor cell lines. The experiments
have demonstrated that the tested drugs; NSC 95397 bortezomib, brefeldin
A, sanguinarine, emetine and CGP-74514A had a considerable apoptotic
effect in the studied human neuroendocrine tumor cells in vitro. At longer
exposure times, these drugs activated the extrinsic apoptotic pathway and at
shorter drug exposure times (24 h), the intrinsic apoptotic pathway resulting
in nuclear condensation and fragmentation.
Hollow Fiber Assay (paper IV)
In paper IV with the in vitro Hollow Fiber, the human atypical bronchial
carcinoid cell line NCI-H720 showed a higher absorbance signal per cell
than typical bronchial carcinoid cell line NCI-H727 when these tumor cells
were cultured inside the hollow fiber for 2 weeks. There was gradual increase in the signals generated by the viable cells throughout the observation
period under the growth condition evaluated. A concentration-dependent
decrease in SI treated with emetine or CGP-74514A was observed for both
cell lines. There was a tendency for the 3-day, high-proliferating cultures to
be more sensitive than the 14-day low-proliferating cultures for emetine (p
<0.05 at 0.2 µM) and for CGP-74514A (p <0.05 at 4.0 µM). Since emetine
and CGP-74514A showed high in vitro activity against the human atypical
bronchial carcinoid cell line NCI-H720, these drugs are possible candidates
for further studies, yet the activity was less towards the human typical bronchial carcinoid cell line NCI-H727. Atypical bronchial carcinoids are more
prone to metastasize and recur than typical ones. It is thus more urgent to
find new active drugs against atypical carcinoids. Another conclusion, from
Paper IV is the confirmation of the possibility to use human tumor cells from
NCI-H727 and NCI-H720 cell lines in the Hollow Fiber assay. This may
hopefully facilitate and reduce the costs for developing new active drugs for
patients with bronchial carcinoids.
31
Future perspectives
Drug discovery is a time-consuming and a labour-intensive process that
evolves through several sequential phases including target identification and
validation, drug design, identification and characterization, clinical candidate
selection and pre-clinical and clinical testing (84). In the development of
novel therapies for cancer treatment it is important to have good insight of
the molecular mechanism of the drug and events involved in the induction of
cell death. The results presented in this thesis have given opportunity to go
further on and do animal studies on these drugs. We are therefore planning
further studies, both in animals and using the Hollow Fiber model, to validate the cytotoxicity of the eleven drugs that have shown cytotoxicity in
vitro. We also aim at performing combination studies with the most interesting drugs and standard chemotherapeutic agents in animals and with the
Hollow fiber model both in vitro and in vivo. We hope that this will lead to
better therapeutic options for patients with neuroendocrine tumors, both by
the synergistic and additive effects per se, by overcoming drug resistance
and by decreasing the side effects of toxic chemotherapeutic drugs, which
may possibly be given in lower doses.
Solid tumors require the formation of new blood vessels when growing to
a size greater than 2 mm in diameter. NETs are highly vascularized and are
also known to express endothelial growth factor receptor (VEGF) (85). The
formation of new blood vessels, called angiogenesis, can be inhibited by
several drugs as human endostatin which inhibits the proliferation and migration of vascular endothelial cells and thereby inhibits angiogenesis and
tumor growth (86).
Bevacizumab (Avastin™, Roche) is a monoclonal antibody against
VEGF with antiangiogenic properties. This drug was compared with pegylated interferon alfa- 2b in 44 patients with carcinoids already taking octreotide. Among the bevacizumab treated patient, four patients (18%) achieved
partial response (PR) and 17 patients (77%) had stable disease (SD). In the
PEGinterferon arm, none had radiological response and 15 patients (68%)
had SD. It is however important to realize that PEGinterferon, which also
has antiangiogenic properties, was given in a lower dose than normally used.
Combinations between antiangiogenic and cytotoxic drugs have also been
tried. In one study with thalidomide, which has antiangiogenic activity by
interfering with the VEGF pathway, and temozolomide, antiangiogenic activity response were observed in patients with endocrine pancreatic carcino32
mas, in carcinoid patients (87). To further optimize the antitumor effect of
the drugs studied in this thesis we would validate the combination between
these drugs and standard antiangiogenic agents, both using the Hollow Fiber
model and in animal experiments. This may hopefully result in new interesting treatment combinations for patients with neuroendocrine tumors, with
increased activity and fewer side effects.
33
Summary of the Thesis in Swedish
Populärvetenskaplig sammanfattning på svenska
Avhandlingen handlar om att med hjälp av olika metoder upptäcka och utvärdera nya substanser som har effekt på neuroendokrina tumörceller. Cancer är en spridd sjukdom och då få patienter botas behövs nya och bättre
cancerbehandlingar. Cancer uppstår då det blir fel i den normala regleringsmekanismen hos skadade celler. I dessa celler uppstår en ohämmad tillväxt
och en tumör bildas. Målet med att hitta bra läkemedelsbehandling är att få
cancercellerna att dö utan att skada normala friska celler. De potentiella cancerläkemedlen studeras noga in vitro, d.v.s. utanför kroppen på celler eller
vävnader innan man går vidare till att studera i djur, och eventuellt människor. Behandling av patienter med lungkarcinoider och endokrina pankreastumörer har gett dålig prognos på tillgängliga medicinska behandlingar som
olika cytostatikakombinationer och bioterapi som alfa-interferon och somatostatinanaloger. För att identifiera och utvärdera lovande nya cytostatika, är
det viktigt att snabbt och enkelt identifiera ämnen med apoptos-inducerande
egenskaper. Under senare år har de molekylära mekanismerna som ansvarar
för apoptos, programmerad cell död, klarlagts. En av de viktigaste grupperna
av proteiner involverade i apoptos är kaspaser, vilka ansvarar direkt eller
indirekt för de morfologiska och biokemiska förändringar som kännetecknar
apoptos. Kaspaser aktiveras av olika toxiska stimuli, vilket leder till apoptos.
Även om patienter med maligna endokrina pankreastumörer samt bronkialkarcinoider som behandlas med cytostatika eller bioterapi, som somatostatinanaloger svarar positivt, uppstår förr eller senare resistens mot behandlingen. Det finns alltså ett behov av bättre behandlingar hos patienter med maligna neuroendokrina tumörer.
Detaljer om ingående delarbeten
Vi har studerat nya cytostatika på neuroendokrina tumörcellinjer genom
singel- eller kombinationsbehandling, baserat på verkningsmekanism, apoptos och cytotoxisk effekt på bronkialkarcinoider; atypisk lungcarcinoid NCIH720 och typisk lungcarcinoid NCI-H727 och endokrina pankreaskarcinoidcellinjen BON-1. Studierna är främst utförda med fluorometrisk microculture cytototoxicity analys (FMCA), där fluorescensen, som är proportionell till
34
antalet levande celler, mäts. Mekanismen för celldöd, apoptos gjordes med
multiparameter apoptos analysen Array Scan HCS.
Karakteriseringen in vitro av den cytotoxiska aktiviteten hos substanserna
studerades med Hollow Fiber assay.
Många av in vitro-modeller som används i preklinisk cancer läkemedelsutveckling bygger på tumörcellinjer. Dessa modeller är tekniskt enkla och
användbara för vidare studier, men de efterliknar inte den komplexa mikromiljö, heterogenitet och proliferativa egenskaper hos solida tumörer, och
detta kan bidra till felaktiga förutsägelser vid in vivo effekter. Läkemedel för
behandling av cancer kan vara effektiv i solida tumörer endast om de kan
tränga in i flera cellager och behålla sin verksamhet i tumörens mikromiljö.
För att lösa detta har tredimensionella in vitro solida tumör modeller utvecklats som in vitro hollow fiber modeller. Den analysen är baserad på att
man odlar tumörceller i ihåliga polyvinylidine fluoride fibrer in vitro eller in
vivo för en levande celltäthet och gör en bedömning av tillväxt och drog
känslighet för tumör cellinjer eller den solida tumören.
Delarbete 1
I delarbete har vi screenat 1280 droger som erhållits från LOPAC1280TM
bibliotek (Sigma Aldrich, St Louis, MO). Läkemedel med överlevnad index
(SI) under 60 % har tagits fram för vidare studier. Studierna utfördes på tre
neuroendokrina tumörcellinjer, som jämförelse studerade vi också en normal
human retinalt pigmentepitel cellinje,; hTERT-RPE1 med fluorometrisk
microculture cytototoxicity analys (FMCA). Den första undersökningen
resulterade i 18 läkemedelskandidater med SI-värde lägre än 60 %. Dessa
läkemedel valdes ut för ytterligare en dos-respons experiment i tre neuroendokrina tumör cellinjer och en normal epitelcellinje. Alla droger testades i
intervallet 0,001 till 100 µM. Den cytotoxiska effekten av föreningarna bestämdes av deras IC50-värden (koncentrationen av läkemedlet där> 50 % av
cellerna dör). Elva av 18 föreningar var känsliga med IC50-värden <10 M.
Sammanfattningsvis har vår studie visat att de elva substanserna, Bay 117085, bortezomib, brefeldin A, CGP-74514A, doxorubicin, emetine, idarubicin, -lapachone, mitoxantron, NSC 95397, och sanguinarine har antitumöreffekt i lungcarcinoid och pankreas carcinoid cellinjer in vitro.
Delarbete 2
I det första delarbetet fann vi 11 föreningar efter en screening på 1280 cytostatika där 50 % av tumör cellerna dog efter behandling av cytostatika med
koncentration <10 M. I delarbete 2 valdes fem av dessa föreningar ut, med
olika verkningsmekanismer, för vidare studier med fluorometrisk microculture cytototoxicity analys (FMCA), som syftar till att utvärdera potentiella
synergistiska effekter när de kombineras med fyra etablerade cytotoxiska
35
läkemedel som redan används i kliniken för behandling av neuroendokrina
tumörer. De fem utvalda föreningarna var brefeldin A, NSC-95397, emetine,
CGP-74514A och sanguinarine klorid. De fyra etablerade läkemedlen var
doxorubicin, etoposid, oxaliplatin och docetaxel. Studierna utfördes på tre
neuroendokrina tumörcellinjer. Kombinationen av de olika cytostatika visade mest synergisk effekt i de neuroendokrina tumörerna, men också additiv
effekt samt en substans visade antagonistisk effekt. Sammanfattningsvis
visar våra resultat att synergiska effekten, men också den additiva effekten
hos de substanser som har studerats är lämliga kandidater för vidare in vivo
studier.
Delarbete 3
I delarbete 3 har vi studerat den mekanistiska effekten apoptos en "skonsam"
programmerad celldöd efter behandling med NSC 95397, brefeldin A, bortezomib och sanguinarine klorid, med multiparameter apoptos analysen Array
Scan HCS. Studierna utfördes på tre neuroendokrina tumörcellinjer. Vid
denna "skonsamma" celldöd monteras cellen ner bit för bit vilket förhindrar
att farliga ämnen i cellen släpps fria. Det finns också en celldöd som kallas
för nekros. Vid nekros spricker cellen och de farliga substanserna läcker ut
vilket leder till att omgivande celler skadas. De flesta anticancermedel som
används idag ger svåra och även dödliga skador på normala celler. De dödar
tumörceller inte bara med apoptos, utan också med nekros. Resultaten visar
att de studerade cytostatika inducerade höga nivåer av kaspase-3 aktivitet
med apoptotiska förändringar, men mindre nekros. Sammanfattningsvis har
våra experiment visat att de testade cytostatika, NSC 95397 bortezomib,
brefeldin A, sanguinarine, haft stor apoptotisk effekt i neuroendokrina tumörceller in vitro.
Delarbete 4
I delarbete 4 har vi studerat den mekanistiska effekten apoptos efter behandling med emetine och CGP-74514A samt karakteriserat den cytotoxiska
aktiviteten hos dessa två substanser med Hollow Fiber assay in vitro. Studierna utfördes på två neuroendokrina tumörcellinjer, atypisk lungcarcinoid
NCI-H720 och typisk lungcarcinoid NCI-H727, emetine och CGP-74514A
inducerade höga nivåer av kaspase-3 aktivitet med mindre nekros. Resultaten med in vitro Hollow Fiber visade att de neuroendokrina tumörerna odlade i fibrer svarade på cytostatika behandlingen. En koncentrationsberoende
ökning av celldöd sågs i båda cellinjerna efter behandling med emetine eller
CGP-74514A. Sammanfattningsvis har våra experiment visat att emetine och
CGP-74514A haft stor apoptotisk effekt i neuroendokrina tumörceller in
vitro. Detta innebär att dessa droger är möjliga kandidater för vidare studier.
Resultaten från denna studie bekräftar också möjligheten att använda mänsk36
liga tumörceller från NCI-H727 och NCI-H720 cellinjer och överföra cellerna till solida tumörer med hjälp av Hollow Fiber analys.
37
Acknowledgements
This work was carried out at the department of Medical Sciences, Endocrine
Oncology and Division of Clinical Pharmacology at Academic Hospital,
Uppsala University. Financial support was provided by grants from the
Lion`s Cancer Research Foundation.
I would like to express my sincere gratitude to colleagues, friends and
family who have contributed to the research and encouraged me in this work
and especially to:
My main supervisor, Docent Dan Granberg, who has guided me with the
essentials and benefits of good medical research throughout the years of my
graduate studies. I am grateful for your patience and understanding of my
life`s restrictions, sorrows and problems during the time as a post-graduate
student. You handled me with encouragement and trust.
Saadia Hassan, co-supervisor, thank you for sharing your time and helping
me with new methods. Thank you for always taking time to read and write in
the manuscripts, for scientific discussion and for all support.
Professor Kjell Öberg, co-supervisor, thank you for believing in me and
giving me the opportunity to work with cancer research at your department.
Henrik Lövborg, previous co-supervisor, I would express my gratitude for
introduce and sharing your experience and knowledge in methods of cytoxicity and for always answer my questions.
Professor of Clinical Cancer Pharmacology, Rolf Larsson I am very grateful
for the opportunity to work and learn new methods in your group.
Professor Eva Tiensuu Jansson, thank you for asking questions, giving me
advice on the Monday meetings and for inviting us to your nice home for
wonderful Lucia celebrations.
Professor Barbro Eriksson because you always took care of administrative
and financial matters. Thank you for your support.
38
Lena Lenhammar, Christina Leek and Åsa Forsberg for your excellent technical support, and help with FMCA, tumor cell lines, chemicals and other
experiments.
Birgitta Sembrant and Annette Hägg for all your enormous help with administrative stuff and with the poster for presentation in Amsterdam. You were
always there when you are needed.
Malin Wickström and Linda Rickardson, my co authors and colleagues for
good collaboration and sharing valuable knowledge about FMCA and Array
Scan to make this thesis possible.
Enrique Vegas for all your help with computers and programs, many good
stories and laughs and for giving me advises to make me belief in life.
I would like to thank past, present and future PhD students, colleagues and
friends at the section of Endocrine Oncology and Division of Clinical Pharmacology: Dr Janet Cunningham, Dr Apostolos Tsolakis, Dr Terese Johansson, Dr Malin Grönberg, Clary Georgantzi, Dr Minghui Liu, Dr Yinghua,
Zhou,Dr Cecile Martijn, Margareta Halin Lejonklou, Dr Camilla Holdstock,
Margareta Ericson, Dr Valeria Giandomenico, Tao Cui, Su-Chen Li and Dr
Daniel Laryea who have created a stimulating and creative atmosphere and
make the departments such a nice place to work in.
All researchers and staff present and past at the Department of Medical
Sciences thanks for creating a friendly environment and for nice coffee and
lunch sessions.
Carin Muhr, thank you for aroused my interest in graduate studies. It is your
credit that I have continued to study for PhD and for travels to Boston and
Reykjavik, for making me feel like a princess in the yellow prom dress, for
your friendship.
My thoughts also goes to other former colleagues at Department of Oncology, Göteborg University, Sahlgrenska University Hospital, thank you for
giving me knowledge in cell culture technology which have been the basis of
this thesis.
Petra Holmström because you always have been such a good friend for so
many years, inviting me to Smögen, “Guldknappen-galan” and much more.
All my friends outside the lab especially Peter A, Marina, Kjell, Anne, Michael, Rodrigo, Paola, Sylvia, Lena, Nicklas, Peter S, Petra for parties
laughter, card games and travelling company.
39
My family in Uppsala for all love and always being there.
My wonderful parents Ingvor and Sune Aronsson for always supporting,
helping and believing in me, for your endless love through life. This thesis is
dedicated to you.
My wonderful son Aron Larsson for bringing endless of laughter and love to
my life.
And finally to Tomas Larsson, my love, thank you for patience and for encouraging me to work harder during the challenging periods and for being
such a wonderful husband.
40
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