Tumor Lung Cancer Model for Assessing Anti-neoplastic Effect of PMF in Rodents: Histopathological Study
This study aimed to elaborate an experimental model of pulmonary carcinogenesis in healthy mice and to ascertain the preventive efficacy of PMF when administered against experimental lung carcinogenesis. Male Swiss Albino mice lineage was carried through an intra-peritoneal injection of the Benzo[a] pyrene diluted in corn oil, a polycyclic aromatic hydrocarbon is widely known by its power of tumoral lung induction. Four experimental groups had been used with 20 animals in each: The first is control group (without infection or treatment); the second is carcinogenic group without treatment, the third is treated carcinogenic group, the fourth is positive control group received only treatment, submitted to euthanasia 08, 16, 24 weeks after the experimental procedure. After 08 weeks, the presence of diffuse inflammatory alterations was observed in carcinogenic- non treated group with thickness of the alveolar wall after the inflammation, however, at analysis of the pulmonary tissue of the treated carcinogenic group it had been observed hyperplasic alterations (BALT hyperplasia) but in positive control group thickness of the alveolar wall was noticed. With more time, after 16, 24 weeks administration of PMF histopathological changes became lesser in the treated carcinogenic group as compared to animals treated with the B[a]P only. In conclusion, the main secondary alterations in the intra-pulmonary instillation of B[a]P of mice were: cellular proliferation, inflammatory alterations of several degrees. PMF treatment has a slightly protective effect to lung tissue along short time but with more time it improved the structure of the lung in carcinogenic treated group.
April 02, 2011; Accepted: May 10, 2011;
Published: July 02, 2011
Annually, more than 5 million people are diagnosed with cancer and more than
3.5 million people die from cancer worldwide (Ferlay et
al., 2000). The management of malignancies in humans still constitutes
a major challenge for contemporary medicine (Coufal et
al., 2007; Widodo et al., 2007). Despite
improvement in therapy, the cure rate for lung cancer remains low. Chemoprevention
offers an important approach to decreasing the incidence of lung cancers. Chemopreventive
agents with strong efficacy against lung cancer often cause systemic toxicities
and adverse effects by standard delivery modalities. Toxicities can often prevent
the clinical use of these agents (Verschraegen et al.,
According to Cragg and Newman (2000), over 50% of the
drugs in clinical trials for anticancer activity were isolated from natural
sources or are related to them. In the last decades a considerable growth in
scientific and medical interest for the use of herbal and traditional medicines
has been observed (Gupta et al., 2001; Liao
et al., 2004). Natural products play an important role in our healthcare
system (Lee, 1999). They offer a valuable source of
potent compounds with wide variety of biological activities and novel structures
and provide important leads for the development of novel drugs (Vuorela
et al., 2004).
Animal studies have shown that green tea, as another natural product, is a
potent inhibitor of lung tumor development (Zhang et
al., 2000). The study of the effects of green tea infusion on the spontaneous
formation of lung tumors and rhabdomyosarcomas in A/J mice demonstrated that
the mice given 1% green tea exhibited a significantly lower lung tumor multiplicity
from 0.72/mouse to 0.41/mouse (Landau et al., 1998).
PM 701 has been discovered to inhibit the growth of lung cancer and leukemic
cells in vitro (Khorsid and Mushref, 2006) and to increase life span
of mice bearing leukemia cells by at least 3 folds which means that it has a
favorable antimitotic effect (Moshref et al., 2006).
Previous investigations of PM 701 (Ahmed et al.,
2009) proved its cytotoxic activity against cancer cells, it caused selective
programmed cell death of cancer cells (apoptosis) of cultured human lung cancer
cells while it had reparative effect on normal human cell (foreskin) (Khorshid
et al., 2005). It was effective in limiting of metastasic spreading
of leukemia cells in animal models (Moshref et al.,
2006). Light and electron microscopic histopathological study was carried
on animal model which proved the reparative effect of this agent (Moshref,
This study aimed to confirm the previous results of present work by created lung cancer animal model using B[a]P then this animal treated by PMF and the results were compared with the results of non treated group.
MATERIALS AND METHODS
The whole project was conducted between 2007-2011 but this specific experiment on animal models was carried on in 2010.
In Tissue culture unit King Fahd Medical Research Center (KFMRC) in King Abdul Aziz University (KAU), Jeddah in Saudi Arabia.
Reagents: PM 701 is a yellowish powdered form, pH 8.3 that has sharp
(offensive) odor and does not soluble in water but has good suspension with
Tween 80 which was stable for at least one month. PM 701 was categorized as
practically non toxic (Khorshid, 2008). PMF is the effective
fraction of PM 701 as proved by El-Shahawy et al.
Benzo[a]pyrene (B[a]P) (99% pure) (a polycyclic aromatic hydrocarbon widely known by its power of tumoral induction) was purchased from Sigma Chemical Co. (St. Louis, MO). B[a]P was prepared immediately before use in animal bioassays by dissolving in corn-oil.
Animal studies: Eighty male Swiss Albino mice lineage (5-6) weeks were obtained from animal house at KFMRC maintained in polypropylene cages of 50x33x20 cm at a constant temperature (21±1°C) and 62% relative humidity. The use of animals was according to the ethical requirements that approved by the Animals Research Ethic Committee of KAU.
Mice lung cancer models in this experiment were carried through an intra-peritoneal
injection of B[a]P diluted in corn oil. Four experimental groups had been formed
with 20 animals each: First control group (no treatment to account for stress
factors during mouse handling procedures affecting tumorigenesis); Second carcinogenic
non treated group (50 mg B[a]P/kg/one time), third treated group (50 mg B[a]P/kg/one
time)+(120 mg PMF/kg/day), fourth positive control group received the treatment
only (120 mg PMF/kg/day), all animals were submitted to euthanasia 2,4,6 months
after the experimental procedure.
Lung tissues were extracted and histological preparation was carried according
to El-Banhawy and El-Gansory (1989). Specimens were fixed
in 10% neutral formalin and the standard procedures of dehydration, clearing
and embedding in wax were followed. The tissue were sectioned at 3-5 μm
and processed for light microscopic investigations adopting the Hematoxylin
and Eosin (H and E) staining procedure (Drury and Wallington,
1980) and submitted to the morphometrical analysis to describe the tissue
In this study, mice cancer models were carried through an intra-peritoneal
injection of B[a]P diluted in corn oil, then a detailed histopathological examination
was conducted to determine the degree of lung tumor progression related to the
effect of PMF on tumor development. The results of the light microscopic investigations
of the carcinogenic lungs after two months, in the group received only B[a]P
suggested that B[a]P is highly toxic; all lung nodules were diagnosed as lung
adenomas (Fig. 1a, b). Where after two months in the B[a]P+PMF-treated
group, the mice lung tissues showed a decrease in tumor load compared to the
B[a]P- group (Fig. 2a, b). After four and six months, all
mice showed great tolerance to treatment with PMF and no clinical evidence of
toxicity was observed as PMF was found to exhibit significant efficacy against
B[a]P-induced mouse lung tumorigenesis with still manage to reveal the type
II pneumocytes that had elongated nucleus with prominent nucleolus and scanty
cytoplasmic contents (Fig. 3a).
||Light photomicrographs representative adenomas from the B
[a] P-group two mouths after the intraperitoneal injection at (a) x100 and
(b) x1000 magnifications
||Light photomicrographs representative efficacy of PMF against
B[a]P-induced mouse lung tumorgensis after two months at (a) x100 and (b)
||(a) Light photomicrographs representative efficacy of PMF
against B[a]P-induced mouse lung tumorigenesis after four months at x400
magnification and (b) Light photomicrographs representative mice were subjected
to PMF treatment for 2 months, note alveolar widened (ulceration) and thick
alveolar septa (arrow). Congested blood vessel (BV) at x100 magnifications
||(a) Light photomicrographs representative mice were subjected
to PMF treatment for 4 months, note metaplasia in the epithelium of the
lung at x100 magnifications and (b) Light photomicrographs of mice were
subjected to PMF treatment for 6 months note normal cytoarchitecture with
well preserved alveoli (arrows) and alveolar septa (arrow-head) at x400
The PMF inhibited tumor load which is commonly interpreted as tumor growth
during tumor progression compared with the group received only B[a]P.
As shown by this experiment the cytotoxicity of PMF well recorded in the group treated only with PMF, where lung congestion and widened alveolar septum, obviously, the first two months are more serious (Fig. 3b), after four months of the treatment the histopathological observation showed an alteration in the cytoarchitecture of the epithelium of the lung of the PMF group, these alterations were characterized by ulceration of the alveoli and desquamation (Fig. 4a). But after six months might be adaptation occur as, the lung tissue cells missing the morphological changes in the structure and the type II pneumocytes keep its normal shape with elongated basophilic nucleus with less prominent nucleolus and scanty acidophilic cytoplasm (Fig. 4b). Whereas, the histopathological observations of carcinogenic animals showed a great deal of alteration in the cytoarchitecture of the epithelium of the lung, these alterations were characterized by tumorgenesis in addition to ulceration in the alveoli and metaplasis which may affect the functional capacity of the studied tissue.
The results of the light microscopic investigations of the carcinogenic lungs
after two months, in the group received only B[a]P suggested that B[a]P is carsinogenetic,
all lung nodules were diagnosed as lung adenomas with agree with Castro
et al. (2008) who mentioned that [B[a]P]-induced mice lung carcinogenesis
without significant systemic toxicity. The histopathological observations of
carcinogenic animals showed a great deal of alteration in the cytoarchitecture
of the epithelium of the lung, these alterations were characterized by tumorgenesis
in addition to ulceration in the alveoli and metaplasis which may affect the
functional capacity of the studied tissue. The mechanism by which B[a]P brought
the ulceration of alveoli was by excavation of the surface epithelium and supporting
tissues of the alveolar wall. The destruction of epithelial cells will diminish
the secretion of surfactant which reduces surface tension within the alveoli
preventing alveolar collapse during respiration. This will in turn reduce the
easy flow of air from the terminal bronchioles in to the alveoli thus, reducing
the alveoli ventilation, as described by Nioya et al.
PMF is a fraction of a natural product, readily available, cheap, sterile and
non-toxic according to chemical and microbiological testing and proved effectiveness
of this agent is reproducible on both in vitro and in vivo models
as previously shown by Khorshid (2008 and 2009)
and Khorshid et al. (2011). In addition to the
previous characters for PMF, we found that after two months of treatment with
PMF the lung tissues showed a decrease in tumor load compared to non treated
group. After four and six months, all mice showed great tolerance to treatment
with PMF and no clinical evidence of toxicity was observed but PMF was found
to exhibit significant efficacy against B[a]P-induced mouse lung tumorigenesis
with still manage to reveal the type II pneumocytes that had elongated nucleus
with prominent nucleolus and scanty cytoplasmic contents. The PMF inhibited
tumor load which is commonly interpreted as tumor growth during tumor progression
compared with the group received only B[a]P.
As shown by this experiment the PMF made few alterations at the first two months in the group treated only with PMF like lung congestion and widened alveolar septum, after four months of the treatment the histopathological observation showed an alteration in the cytoarchitecture of the epithelium of the lung of the PMF group like ulceration of the alveoli and desquamation. But after six months might adaptation occur as, the lung tissue cells missing the morphological changes in the structure and the type II pneumocytes keep its normal shape with elongated basophilic nucleus, less prominent nucleolus and scanty acidophilic cytoplasm.
Previous results of cells count experiments showed severe drop of human lung
cancer (A549) cells number when incubated in PMF compared with the number of
control cells (cancer non-treated) that incubated in MEM media. The activity
of PMF appeared here is due to the antiproliferative effect and apoptotic effect
of this substance on different cancer cells as shown by Khorshid
et al. (2009). It is likely that some degree of apoptosis and inhibition
of cell proliferation might contribute to decreases in tumor load as occurred
in present experiment where PMF might contribute to decreases in tumor load
along six months, some of these results were described by Mahboub
and Khorshid (2010) where they investigated the role of green tea extract
on the proliferation of human ovarian cancer cells.
PMF as extracted from PM 701 contains cupper and Zn as elemental analysis and
some amino acids as theronine, cysteine, tyrosine and methionine, also contains
S-Methylglutathione as mentioned by El-Shahawy et al.
(2010). Many previous studies may contributed to present findings here by
explaining the role of Zn as an essential trace mineral that plays a key role
in many important body processes such as binding DNA and RNA producing energy,
regulating the immune system and cell metabolism. Zn as antioxidant that blocks
the action of activated oxygen atoms which are known as free radicals and can
damage cells (Galan et al., 2005). Other studies
indicated that Zn affects various enzymes and transcription factors which are
important for normal cell proliferation and differentiation; it modulates DNA
replication, protein synthesis and cellular signaling pathways which described
by Khorshid (2004) and Wong and
Abu Baker (2008).
Also Cup and Zn elements are essential for several biological functions throughout
life such as repairing cells and protecting them from damage as mentioned by
Sandstead and William (2007). Copper is a trace mineral
that is needed for many important body processes. Animal studies have shown
that copper is useful in maintaining antioxidant defenses that block the action
of activated oxygen atoms which are known as free radical and can damage cells
(Araya et al., 2005). Also it protects rat liver
from cancer damage and the intake increase of copper has been found to reduce
the occurrence of cancer in the test of animals (Coates
et al., 1989). All these studies explained the reparative effects
of PMF in treated mice, where PMF contains Zn and Cu.
In addition the PMF contains some amino acids as theronine, cysteine, tyrosine
and methionine which are very important for damage the proliferated cancer cells
as stated by Vuorela et al. (2004) suggested
the presence of both peptide and receptor has been found to bind OGFr (opiod
growth factor) and hence a reduction in OGFr-OGFr interactions that would repress
cell replication. S-Methylglutathione in PMF extracted content acts as an important
defense mechanism against certain toxic compounds such as drugs and carcinogens,
these results were consistent with Shimizu et al.
In the present study, PMF was the target for its anticancer effectiveness as it was capable of killing cancer cells. Animal model (mice) for cancer was induced chemically using Benzo pyerine given to animal via IP. The success in achieving metastasing lung cancer was evident. Also, the safety of PMF was proved here, where control group was also included for finding any side effects. The results were encouraging, animal survival was more in treated group. Histopathological examination of the affected tissues showed marked deceased in tumor size in lung as inducing apoptosis was proved to be the main effect of the treatment.
The authors gratefully acknowledged financial support of El-Zamel's scientific chair, Research and consultation institute, King Abdulaziz University.
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