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Introduction:
Neoplasms are the second leading cause of death after vessel diseases, despite the huge
progress in medical sciences in the last 20 years. Recently, gastric cancer morbidity has
decreased, but mortality is still at high level (Partyka et al., 2012). It considered to be the fourth
most common cancer and the second most frequent cause of cancer-related death, accounting for
10.4% of cancer death worldwide (Amedei et al., 2011). Also, colorectal cancer represents the
fourth commonest malignancy, and constitutes a major cause of significant morbidity and
mortality among other diseases (Zhang et al., 2012). More than 1.2 million new cases of
colorectal cancer are reported each year worldwide (Bünger et al., 2012).
Despite the improvements, estimated cure rates for patients with advanced stages of cancer
remain poor. Therefore, it is important to understand the nature of these tumors to investigate
new therapeutic strategies for treatments (Amedei et al., 2011). Moreover, innovative screening
tools could aid to detect cancer at early stages and allow curative treatment interventions
((Bünger et al., 2012).
Pathological angiogenesis is a hallmark of cancer and various ischemic and inflammatory
diseases. Solid tumors progression largely depends on vascularization and angiogenesis
(Chekhonin et al., 2012). Lake of thoroughly validated predictive biomarkers has been one of
the major hurdles to stratify cancer patients and to monitor tumor progression and response to
therapy. Concentrated efforts in this area of research are leading to discovery of a growing
number of pro- and anti-angiogenic molecules. This integrated understanding is leading to the
development of a number of exciting approaches to treat cancer as antiangiogenic therapy
(Carmeliet and Jain, 2000 and Shojaei, 2012).
Angiogenesis is the result of an imbalance between positive and negative angiogenic factors
released by tumor and host cells into the microenvironment of the neoplastic tissue. Tumor cells
and stromal cells produce various angiogenic factors, including, vascular endothelial growth
factor (VEGF), platelet-derived endothelial cell growth factor (PD-ECGF), cysteine cathepsinsB, interleukin-8 (IL-8), hyaluronic (HA) and nitric oxide (NO) (Kitadai, 2010 and Miyamoto et
al., 2011).
VEGF is a potent angiogenic agent that acts as a specific mitogen for vascular endothelial cell
through specific cell surface receptors (VEGFR). Pathways involved in angiogenesis include
VEGF-A binding to VEGFR-2, VEGF-A, VEGF-B binding to VEGFR-1 and VEGF-C and
VEGF-D binding to VEGFR-2 and VEGFR-3. Other pathways include angiopoietin/Tie,
hypoxia-inducible factor and integrin pathways (Sun, 2012). VEGF suppression leads to
retrogression of neoplastic vessels and tumor growth restriction (Chekhonin et al., 2012).
PD-ECGF is a non glycosylated single chain polypeptide that stimulates chemotaxis for
endothelial cells in vitro and angiogenesis in vivo and plays a direct role in promoting
lymphangiogenesis and metastatic spread to lymph nodes (Kodama et al., 2010). PD-ECGF and
its receptor expression had been reported in a variety of cancer but the role of their expression in
the development and progression of cancer has not yet been elucidated (Kitadai et al., 2006).
Cathepsins are lysosomal proteinases that associated with tumor invasion and metastasis.
Elevated expressions of cathepsins and diminished levels of their inhibitors have been observed
in several human cancers. It have been suggested to be biological markers of malignant tumors
and have proved useful for prognosis of the disease and may be a potential target for cancer
therapy (Nomura and Katunuma, 2005 and kuester et al., 2008).
IL-8, a ckemokine with a defining CXC amino acid motif, is known to possess tumorigenic
and proangiogenic properties. Over-expression of IL-8 has been detected in many human tumors
and associated with tumor growth, metastasis, chemoresistance, angiogenesis and poor
prognosis, implying IL-8 to be an important therapeutic target (Ning et al., 2011 and Ju et al
2012).
Hyaluoronic acid (HA) is a major macropolysaccharide in the extracellular matrix of connective
tissues, is intimately involved in the biology of cancer, It accumulates into the various human
tumors and modulates intracellular signaling pathways, cell proliferation, motility, invasion,
tumor growth and metastasis. High levels associated with poorly differentiated tumors and
aggressive clinical behavior. HA have also therapeutic implications since it is involved in
multidrug resistance (Sironen et al., 2011).
Nitric oxide (NO) is involved in a number of physiological and pathological processes. As an
important biological mediator, NO has been the focus of cancer study for its function in
tumorigenesis, tumor progression and death. The effects of NO on tumor cells are multifaceted,
but many details underlying these effects are not yet well understood (Sang et al., 2011). The
levels of NO critically, modulates the recognition of the tumor cells by dendritic cells, and
proposes a new potential therapeutic approach against chemo-immunoresistant tumours
(Kopecka et al., 2011).
We hypothesized the changes in serum levels (VEGF, PD-ECGF, Cathpsin-B, HA, IL-8 and
NO across the spectrum of gastric-colocrectal cancer and their relationships to the pathogenesis
of cancer. We also clarified the consequence of estimating these indices in untimely tumor
recognitions.
Material and Methods:
The case control study includes 30 patients with gastro-intestinal malignancy who were
selected from South Egypt Cancer Institute in Assiut University, Assiut, Egypt. They were
divided into 4 groups according to the type of the tumor: Group I: patients with gastric cancer
(n=7), group II: patients with colorectal cancer (n=8), group III with anal cancer (n=10) and
group VI (n=5) with other type of GIT cancer as pancreatic and gall bladder cancer.
In addition, there were 10 patients with benign gastro-intestinal mass. Also, 20 healthy
controls of matched age and sex were matched from subjects attended to annual physical check
up without evidence of any cancer.
Complete medical history, physical examination, and routine investigations were done for all
participants. All patients subjected to abdominal computer tomography, magnetic resonance
imaging and upper and lower endoscopy when required. The GIT cancer was classified
according TNM staging system (Sobin et al., 2009). In addition they were classified according
to the size of cancer into high tumor burden (>6x6 cm) and low tumor burden (≤6x6 cm).
Primary tumor in other organs, hereditary cancer syndrome, previous malignancy, previous
radiotherapy or chemotherapy were excluded.
The study was approved by the ethics committee of Faculty of Medicine, Assiut University,
Egypt and informed consents were obtained from each patients and controls.
Fasting 10 ml of blood sample were collected by vein puncture under complete aseptic
condition. Samples were allowed to clot for 30 minutes and centrifuged at 1000 g for 10 minutes.
Serum was collected and stored at -70 ºC in aliquots till time of assay.
Serum VEGF levels were measured with Qauantikine ELISA Kite, (Cat. no. DVEOO, R and
D system, Minneapolis, MN), according to the method of He et al. (1999). Serum PD-ECGF
levels were determined chemically according to the method of Scocca, (1978). Also, serum
cathepsin-B levels were determined chemically according to the method of Barrett, (1970) and
(1972). Serum levels of IL-8 were measured with ELISA kite (cat. No. 2237, Immunotech,
France). Serum levels of hyaluronic acid were determined chemically by the methods of
Greiling, (1961) and NO levels were determined calorimetrically as total nitrite and nitrate as
described by van Bezooijen et al. (1998).
Statistical analysis:
Following application of the Shapiro-Wilkes test to determine a normal distribution, noncategorical data distributed normally are expressed as mean (standard deviation) and data distributed
non-normally are expressed as median (interquartile range). Categorical data are analysed by the chisquared test. Continuously variable data are analysed by ANOVA or the Kruskal-Wallis test.
Tukey’s post-hoc test was used to determine differences between group (data were log transformed if
necessary).
Correlations were required by Spearman’s rank method. A probability of less than 0.05 was
considered as statistically significant. Analyses were done using Minitab Version 14
(Minitab Inc., State College, Philidelphia, PA. USA )
Results:
The demographic data of patients and controls were clarified in table (1).There were no
significant difference as regard to the age and sex. Serum levels of VEGF, PD-ECGF, IL-8, and
NO were significantly higher in benign group as compared with control group (p< 0.0001, p<
0.0001, p< 0.0001 and p<0.05 respectively) and all biomarkers were significantly higher in
cancer group as compared with control and benign group (p < 0.0001 for each) Table (2).
The serum levels of VEGF, PD-ECGF, cathpsin-B, IL-8, HA and NO were significantly
elevated in stage IV and III as compared with stage I and II (P < 0.001, P <0.001, P<0.01,
P<0.001, P < 0.0001 and P < 0.05 respectively) Table (3). As regard to the anatomical type of the
tumor, the serum levels of all biochemical parameters show no significant difference between
gastric, colorectal and anal carcinoma (P> 0.05) Table (4).
In addition, there were significant elevation of the serum levels of VEGF, PD-ECGF, IL-8
and NO, in patient with high tumor burden as compared with patients with low tumor burden (P<
0.05 for each) Table (5). The correlation between various biochemical parameters in cancer
patients was shown in table (6). There was significant positive correlation between the serum
levels of VEGF, PD-ECGF, cathepsin-B, IL-8, HA and nitric oxide. Also, there was significant
positive correlation between the above biochemical parameters and stage of tumors. On the other
hand, there was no significant correlation between these parameter and patients with high or low
burden tumors.
Discussion:
Pathological angiogenesis is a hallmark of cancer and various ischaemic and inflammatory
diseases. Concentrated efforts in this area of research are leading to discovery of a growing
number of pro- and anti- angiogenic molecules. The complex interaction among these molecules
are now beginning to be elucidated and understanding this integration is leading to the
development of a number of exciting and bold approaches to treat cancer (Carmeliet and Jain,
2012). The aim of the present study was to evaluate the angiogenic factors (VEGF, PD-ECGF,
cathpsines, IL-8, HA and NO) in patients with gastric and colorectal cancer and determine their
relationship with the clinico-pathological parameters, early tumor detection and targeted tumor
therapy by using antiangiogenic therapy which thought to be a promising approach to cure
cancer.
In the present study, serum levels of VEGF were significantly increased in all GIT
malignant tumors in comparison with controls and benign tumors group. Moreover, its levels
being significantly higher in stage IV and III versus stage I and II and in patients with high tumor
burden versus low tumor burden. In agreement of the present data, Karayiannakis et al.(2002);
Ohta et al. (2003), Ding et al. (2005), Al-Moundhri et al. (2008), and Halmaciu et al. (2012)
reported higher levels of VEGF in patients with gastric cancer than in healthy controls.
According to Karayiannakis et al. (2002), VEGF serum levels correlated with tumor grade and
size in accordance with our findings. However, Al-Moundhri et al. (2008), could not find any
correlation between VEGF levels and clinico-pathological features, in the form of TNM staging
as well as differentiation.
In addition, De Vita et al. (2004) and Kwon et al. (2010) reported that serum levels of
VEGF were significantly higher in patients with colorectal cancer than controls. Moreover, De
Vita et al. (2004) found that VEGF- and its receptors are expressed at high levels in metastatic
human colon carcinomas. Consequently, VEGF is recognized as a prominent angiogenic factor
in colon carcinoma and the assessment of VEGF expression may be useful for predicting
metastasis (Cascinu et al., 2000) and high levels of VEGF expression were associated with
advanced cancer stage and related to unfavorable prognosis (Cao et al.; 2009 and Liang et al;
2010). Morales-Gutierrez et al. (2011) added that, VEGF expression was significantly higher in
tumor area than non tumor area and this levels reflected tumor stage and used as a poor
prognostic parameter. Also, Li et al. (2012) and Villarejo-Campos et al. (2012) added that
preoperative serum VEGF may be used for evaluating the biological behavior, invasion and
metastasis of gastric and colorectal carcinoma and may help for identify patients with poor
prognosis.
In the present study, serum levels of PD-CEGF were significantly increased in all GIT
malignant tumors in comparison with controls and benign tumors group. Moreover, its levels
being significantly higher in stage IV and III versus stage I and II and in patients with high tumor
burden versus low tumor burden. In our study ,the mean levels of this factor in cancer patients
was more than triple the normal and more than double levels in patients with benign lesions.
These results were in agreement with Kandil et al. (2004) who reported that PD-ECGF was
significantly higher in malignant than control group and the expression was more in the stromal
cells than tumor cells. They added that, the increased expression was present with increase in
depth of invasion. This could be attributed to the insufficient perfusion to areas of the tumor
resulting in the existence of hypoxia, glucose depletion and low Ph which might be involved in
the higher amounts of PD/ECGF. In their histochemical evaluation of the source of this factor,
Kandil et al. (2004) reported a significant correlation was observed between tumor and stromal
expression of PD/ECGF .This finding suggests that stromal cells expression of this angiogenetic
factors, plays an important role in angiogenesis and progression of gastric carcinoma.
Konno et al. (2001), found that, the expression of PD/ECGF in gastric carcinoma can
promote tumor growth and Osaki et al. (2000) found that increased PD/ECGF expression is
closely related with decreased apoptosis in gastric carcinoma. Kodama et al., (2010) added that,
secretion of PDGF by gastric carcinoma cells and expression of its receptors by tumor-associated
stromal cells are associated with lymphatic metastasis so blocked of PDGf receptors signaling
pathway may inhibit node metastasis in gastric cancer.
In case of colorectal carcinoma, Taketayashi et al., (1996) reported that, there was
increased PD/ECGF expression in colorectal tumor tissue compared to normal tissue Moreover,
high PD/ECGF levels are a prognostic factor for poor survival in colorectal cancers (De Brain et
al., 2006). Using staining techniques PD/ECGF is often over-expressed in tumor infiltrating
cells, mainly macrophages, and colon cancer cells themselves (Takahashi et al., 1996 and Van
Triest et al., 2000). According to Temmink et al.(2007), PD/ECGF expression in human
colorectal tumors by infiltrating cells provides an additional alternate mechanism for tumor
neovascularization.
In the present study, serum levels of Cathepsin-B were significantly increased in all GIT
malignant tumors in comparison with controls and benign tumors group. Moreover, its levels
being significantly higher in stage IV and III versus stage I and II but there was no significant
difference in its levels in patients with high tumor burden versus patients with low tumor burden.
These results were in agreement with Hiran et al. (1993) who suggested that serum and
urinary cathepsin-B levels may possible indicators for GIT malignancies and for detecting
remote metastasis. Also, Herszenyi et al. (2000) reported that cathepsin-B was significantly
higher in homogenates of gastric cancer tissue than in those of cancer-free samples obtained
from the same stomach, clearly indicated that this enzyme is involved in development and
growth of this disease. In addition, Russo et al. (2000) found that, cathepsin-B significantly
correlated with TNM stage, nodal status, histological grade and presence of metastasis and
patients with poorly differentiated tumors, indicating that the synthesis and release of cathepsinB is related to bad prognosis.
Similar to present findings, Jevnikar et al, (2008) found that cathepsin-B protein and activity
levels have been found to be higher in both gastric and colorectal cancer. Also, Del Re et al.
(2000) reported that higher activities of cathepsin-B have been observed in the initial stages of
cancer development and higher levels of cathepsin-B have been detected in metastatic versus
non-metastatic cancer tissue homogenates. Moreover, Chan et al.(2000),
observed that
cathepsin-B in the vast majority of human colon adenoma and the cancers, independent of the
stage and its expression was significantly associated with an increased risk of colon cancerspecific and overall mortality. Cathepsin-B was not associated with other molecular features of
colon cancer except for the presence of KRAS and BRAF mutations, thus supporting the
potential for cathepsin-B as a target for molecular detection of neoplasia and therapeutic
intervention (Chan et al.;2000). They found that, that synthesis and secretion of cathepsin-B is
increased in the extracellular compartment of colon cancer so it plays an essential role in
disrupting the exracellular matrix barriers between tumors and surrounding tissues thereby
facilitating invasion and metastasis.
Nomura and Katunuma, (2005) studied the involvement of cathepsin in the invasion,
metastasis and proliferation of cancer cells. Their results indicated that cancer cells orchestrate
various cathepsins to progress malignant disease and it may be a potential target for cancer
therapy. In addition, Miyamoto et al. (2011) concluded that, cathepsin is highly expressed in
GIT tumors compared to its expression in other cancerous lesions, this identifies catepsins as
new diagnostic marker for GIT tumors.
In the present study, serum levels of IL-8 were significantly increased in all GIT malignant
tumors in comparison with controls and benign tumors group. Moreover, its levels being
significantly higher in stage IV and III versus stage I and II and in patients with high tumor
burden versus low tumor burden.
In agreement with present findings, Kitadai et al. (1998) studied expression of IL-8 in human
gastric cancer and found that most tumor tissues express IL-8 at levels higher than those in the
corresponding normal mucosa. The IL-8 mRNA level in neoplasms correlates strongly with
vascularization, suggesting that IL-8 produced by tumor cells regulates neovascularization.
Moreover, Kido et al., (2001) reported that, gastric specimens have increased IL-8 protein levels
and many gastric cancer cell lines express high levels of IL-8 mRNA and protein.
In addition, Kitadai et al. (2000) found that gastric cancer cells express not only IL-8, but
also its receptors and IL -8 treatment increases the invasive capacity of gastric cancer cells and
that IL-8 may have autocrine/paracrine roles in the progressive growth of human gastric cancer.
Moreover, Ju et al. (2012), Kuai et al. (2012) and Ning et al. (2012) concluded that, overexpression of IL-8 promotes the adhesion, migration, invasion and chemoresistance of gastric
cancer cells, indicating that IL-8 is an important therapeutic target in gastric cancer.
Terada et al. (2005) investigated the relationship of IL-8 in colorectal carcinoma and its
levels were significantly higher than those normal tissues. The IL-8 level was significantly
associated with tumor size, depth of infiltration, stages and liver metastases so it was responsible
for tumor progression. According to Rubie et al. (2007) significant IL-8 upregulation was
demonstrated in all inflammatory non-malignant and malignant colorectal entities compared to
their corresponding normal tissues. However, the magnitude of IL-8 expression in surgical CRC
tissue specimens correlates with increasing tumor stages and with the malignant status of
colorectal cancer cells. Moreover, they added that, significant IL-8 over-expression was found in
CRC liver metastasis in comparison to related primary colorectal tumors.
According to the study of Rubie and Colleagues (2007) and Lee et al. (2012), they found a
correlation between IL-8 expression and the development of liver metastasis in colorectal cancer
so, monitoring the IL-8 expression may potentially help to assess the course of cancerous
conditions and its prognosis. Moreover, Nastase et al. (2011) concluded that, IL-8 consist in a
valuable panel of biomarkers, whose detection can be used in early detection and prognosis of
colon cancer. Also, Bünger et al. (2012) reported that, the screening value of additional blood
markers, as IL-8, and the potential advantage of combining serum biochip testing with fecal
occult blood needs to offers an innovative approach to colorectal cancer screening.
In the present study we determined the serum levels of NO in patients with gastrointestinal
tumors. The levels in patients with benign tumors were significantly increased compared with
healthy controls. In patients with cancer, the levels were significantly higher than healthy
controls and benign tumors. In patients with GIT tumors, patients having advanced stage or high
tumor burden showed significantly higher NO levels compared to those with early stage and low
burden. The present findings were in agreement with previous studies which showed that, NO
activity and NO synthesis were high in GIT tumor tissue and in plasma (Szaleczky et al., 2000,
Haklar et al. 2001, and Bakan et al., 2002). Also, Bakan et al. (2002) reported higher levels of
NO in the form of nitrate and nitrites as the stage of the disease increased.
In addition, Kishikawa et al. (2011) reported that, measuring the serum nitrate/nitrite
concentration has potential in estimating the gastric juice nitrate/ nitrite concentration which
could be useful as a marker for mutagenesis. Also, Zhang et al. (2011) concluded that, inducible
nitric oxide synthase expression correlates with lymph node metastasis, vascular invasion, distant
metastasis, TNM stage and poor survival rate in gastric cancer. They propose that synthesized
inducible nitric oxide synthase increases angiogenesis, and lymphangiogenesis thus promotes
tumor progression. Inducible NO synthase expression may be a good biomarker for poor
prognosis in gastric cancer (Zhang et al., 2011).
Kopecka et al. (2011) added that, the levels of NO critically modulate the recognition of
tumor cells by dendritic cells, and proposes a new potential therapeutic approach against chemoimmunoresistant tumors. On the other hand, Sang et al. (2010) and Sang et al. (2011) found
that, NO not only inhibit cell growth and proliferation, but also induces apoptosis in gastric
cancer and such effects of NO showed significant dose-dependant activity.
The apparent paradox, in which NO both enhances and inhibits tumorigenesis, has been
attributed to several factors including local NO concentrations, cell type, cellugenetics, and
redox status (Yoshie and Ohshima,1997). Low levels of NO appear to increase the tumor –
promoting effects of NO, whereas high levels are cytostatic/cytotoxic. iNOS activity in
genetically engineered human colon cells with increased growth and angiogentic potential is as
least 1-2 orders of magnitude lower than that associated with anti-tumor actions such as nectotic
and apoptotic cells death (Jenkin et al.,1995).
NO is involved in many of the patho-physiological processes linking inflammation to cancer
development and progression in the GIT tumors. When the generation of NO exceed the antioxidation capacity of the cell NO and its reactive nitrogen oxide species are capable of damaging
DNA and proteins. In particular, NO-mediated nitrosylation of DNA repair proteins inhibits their
enzymatic activity, potentiating accumulation of DNA damage. NO can also nitrosylate and
inactivate the effectors proteins of apoptosis, especially caspases. Inhibition of caspases resulting
in dysregulation of apoptosis, further promoting malignant transformation (Jaiswal et al., 2001).
In the present study, the levels of HA were significantly increased in patients with GIT cancer
compared with either healthy controls or patients with benign tumors. Its levels were higher in
stage IV and III versus stage I and II, but there was no significant difference between patients
with high and low tumor burden. These findings were in agreement with Vizsos et al., (2004)
and Aghcheli et al., (2012) who reported significantly elevated levels of hyaluronic acid in
gastric cancer patients and its serum levels showed fairly good sensitivity for discrimination of
gastric cancer patients from controls. Also, Misra et al. (2008) reported significant increase of
hyaluronan in colon cancer.
In addition, Tammi et al. (2008), reported that, two different patterns have been suggested
for expression of hyaluronic acid in tumors arising from simple and stratified epithelia. With the
first group, such as cancers of the colon and stomach, the expression correlates with tumor grade
and invasiveness. With poorly differentiated tumors and local or distant invasion, more cancer
cells will be hyalyronan-positive or have more intense hyaluronan staining. On the other hand,
early stage cancer arising from stratified epithelia may have increased hyaluronan expression
compared to normal epithelium, but high-grade aggressive squamous cancers are associated with
decrease in hyaluronic acid expression Aghcheli et al. (2012).
Not only HA but also its receptors as, CD44 and HA- mediated motility, expression has been
positively correlated to metastasis (Ouhtit et al., 2007). The NH2-terminal function area of
CD44 can join the hyaluronate in the basement membrane to extracellular matrix, thus to
regulate the movement and function of cells. By this mechanism neoplastic cells adhere itself to
the extracellular matrix and basement membrane of the host cell, resulting in invasion and
metastasis of malignancy. Also, the degraded products of HA can motivate the growth of local
vessels providing the basis for invasion and metastasis (Chen and Wang, 2000). Moreover,
Sumiya et al. (2011) reported that, the intracellular HA binding protein related to the invasion
and metastasis through promotion of cancer cell motility through a number of pathways
including focal adhesion kinase and MAP kinase. HA derived from malignant cells educates
neutrophils to adopt an activated phenotype, and in that way stimulates the metastasis of
malignant cells, which represents a positive regulatory loop between tumor and their stroma
during neoplastic progression (Wu et al., 2011).
In the present study, there were positive correlations between different studied indices. These
results were in agreement with Brown et al. (2000) who suggested that thymidine catabolism by
PD-ECGF stimulate carcinoma cell secretion of IL-8 and VEGF. Also, Sandhu et al. (2000),
reported the role of inflammatory cells infiltrating tumor which explain the presence of
chemotectic factor as IL-8 that identified in human gastric and colorectal cancer. Hypoxic
vascular endothelial cells may be another source of IL-8 which explain the positive correlation
between NO and IL-8.
Bünger et al. (2011) reported that, the assessment of serum levels of IL-8, VEGF, and other
angiogenic factors could be useful for GIT cancer screening with the potential of also detecting
early stage tumor. Bünger et al. (2011) and Büngar et al. (2012) found that, the usage of
additional markers on a multiplex array formats, utilizing serum samples, offers an innovative
approach for cancer screening.
In conclusion, the results of the present study suggested that the angiogenic biomarkers as
VEGF, PD-ECGF, cathepsin-B, IL-8, HA and NO increased markedly in patients with gastric,
colorectal and anal cancers. The increased levels were observed in late stage of cancer and in
patients with high tumor burden. Accordingly, estimation of these biomarkers may used in early
tumor detection and targeted tumor therapy by using anti-angiogenic therapy which thought to be
a promising approach to cure cancer.