paper - Osiris

Background and introduction
Globally, Lung cancer is one of the most common and deadliest forms of cancer with
about 1.35 million deaths from 10.9 million new cases. It also contributes to about 1.18 million
out of 6.7 million cancer related deaths (Okur et al, 2012). Non small cell cancer (NSCLC) is
responsible for about 80% of all lung cancer cases. NSCLC cancer happens to be one of the most
common forms of lung cancer. It grows relatively more slowly when compared to small cell lung
cancer (SCLC). This cancer affects varying types of epithelial tissue in the lungs. The most
common are squamous cell carcinoma which usually occurs in the bronchus, large cell
carcinomas which occur in any part of the lungs and adenocarcinomas which manifest in the
outside area of the lungs (Zhu et al, 2012). The principal cause of this cancer is smoking, with
varying degrees of risk depending on longetivity and frequency of smoking. Other risk factors
may also include second hand smoking, and exposure to carcinogenic chemicals such as
asbestos, uranium, beryllium and coal products. Common symptoms might be chest pain, blood
cough, bone pain and weakness. Depending on the tumour stage and patient characteristics
common treatment options are surgery, chemotherapy, radiotherapy and a combination of
these options (Okur et al, 2012). Furthermore, the most fundamental factor in survival is based
on early detection. According studies carried out by Okur et al. (2012), on 238 lung cancer
patients , two year survival for stage 1 was 84 % as opposed to 34% and 9 % for stages 3 and 4
Classic lung cancer drugs such as cisplatin , carboplatin, accompanied by new ones such
docetaxel and vinorelbine has always been used in treatment of lung cancer patients but the
adverse side effects of hair loss, allergic reactions and most worryingly chemo resistance seems
to be a big problem up till date. Buffalin is a toxic steroid with anti – tumour effects that is
derived from a Chinese medicine called chan`su (Zhu et al, 2012). Bufallin induces apoptosis
and slows down the growth rate of leukemia, gastric, colon and breast cancer. According to
studies carried out by Xie et al. (2012) buffalin even induces autophagy mediated cell death in
human colon cancer by activation of the JNK pathway. It also triggers apoptosis in human T4
cancerous bladder cells by arresting cells in both the Go and G1 phases by decreasing the levels
of pro proliferative agents like cyclin D , cyclin E and CDK 2 and 4 (Huang et al, 2012). However,
with the all these anti tumour characteristics of buffalin, it had never been demonstrated in
lung cancer until the findings and publications of Zhu et al in 2012. In the work carried out by
Zhu et al, the A549 human lung adernocarcinoma epithelial cells were used. They found that
PI3K /Akt pathways regulated apoptosis of tumour cells and aided their proliferation and
chemo resistance. To that effect, they hypothesised that buffalin could induce the apoptosis of
lung cancer through the regulation of the PI3k/ Akt pathways.
Results and methodology
In other to prove that bufallin induces apoptosis and promotes proliferation, MTT assay
was used to test the effects of buffalin on cancerous A 549 cells. These cells where treated for
48, 72 and 96 hrs and the results can be seen of figure 1 on the appendix below. From the
figure, one can clearly see that cell growth was inhibited by bufalin in a dose and time
dependent manner. That’s to say the longer the cells stay in contact with the drug, the lesser
they proliferated and the higher the dose of the buffallin, the higher the inhibition of growth.
The next thing researchers wanted to find out was the actual mechanism behind this
inhibition of growth, so they turned to propidium iodide (PI) and the flow cytometry techniques
in other to find out if apoptosis was behind this under proliferation. For 48 and 72 hrs, A 549
cells where treated with 20, 40 and 100nmol/l of buffalin. PI was carried out, followed by flow
cytometry and the results can be seen on figure 2 on the appendix below. From the figure, one
can see that buffalin is inducing apoptosis on the A 549 cells in a gradual time and dose
dependent manner because of the gradual decrease in the number of cells (apoptosis) as the
dose and time in contact with drug increases.
They also went further to stastically quantify the rate of apoptosis by showing the
difference between cells treated with buffallin and control cells without treatment. Looking at
the results shown in figure 3, one can see that there is a significant difference (p < 0.01)
between cells treated with buffalin and those without it because those treated underwent
apoptosis in a dose and time dependent manner while the control did not.
After they found out that it was apoptosis that was responsible for the regulated cell
growth they then went forth to observe the morphological changes associated with a cell that
undergoes apoptosis. As seen in figure 4, using giemsa staining under a 200x magnification,
they could clearly see the morphological changes of cytoplasmic shrinkage, nuclear
condensation and apoptotic bodies that are usually associated with apoptosis. To then prove
that buffalin decreases anti – apoptotic proteins and increases pro-apoptotic proteins,
western blot analysis was performed on A549 cells treated with 100nmol/l of bufallin for 12,
14 and 48hrs. The results as seen on figure 5 below shows that buffalin increases the
expression of bax (pro apoptotic) and decreases the expression of bcl-2(anti apoptotic) in a
time dependent manner. Furthermore, same western blot analysis was used to find out the
different levels of cleaved caspase 3 and pro caspase 3 and as seen from the results in figure 6
below, prolongation of buffalin treatment increased the levels of cleaved caspase 3. To
confirm their new finding that buffalin increases activation of caspase 3, a caspase 3 inhibitor
known as Z-DEVD-FMK was used in combination buffalin on the A 549 cells. As shown from
the results in figure 7 from the appendix below, percentage of apoptosis decreases when the
inhibitor is mixed with buffalin compared to when buffalin is used alone, showing that buffalin
regulates caspase 3 activation. Finally, to prove essentiality of the inhibition of Akt pathway
in order to induce apoptosis, pre-treatment of the cells with an an Akt inhibitor known as
LY294002 was carried out. As shown in figure 8 below, with the help of flow cytometry, it was
found that LY294002 synergizes with buffalin treatment to induce apoptosis. And as seen in
figure 9, Western blot analysis also revealed that pretreatment with ly294002 worked with
buffalin treatment to upregulate bax and caspase -3 while down regulating bcl-2 and livin
thus they could accurately conclude and confirm their hypothesis that bufalin inhibits the
p13k and Akt survival pathways in order to induce apoptosis in lung adernocarcinoma cells
From the findings of these researchers, one can clearly see that buffalin induces
apoptosis in lung cancer cells by inhibiting the activation of the PI3k/ Akt pathway. This could
be very applicable in that it could be used in combination with chemotherapy to better combat
chemo resistance in lung cancers. However, researchers failed to show the exact mechanism of
action by which bufalin inhibits the Akt pathway. This means that we don’t know exactly what
molecules are affected in the Akt pathway. The closest the researchers got was discovering that
bufalin treatment reduced phosphorylated levels of Akt when compared to control cells but
they couldn’t tell exactly why and what molecular interactions were involved. The exact
mechanism of action might be very important in developing drugs with not only buffalin as an
active ingredient but other derivatives and even better versions due to the complicated and
adaptive nature of cancerous cells.
To conclude, lung cancer is the still the leading cause of deaths related to cancer with
about 226 000 new cases and 160 000 deaths in the United States this 2012 (MacConail 2012).
However, projections show this numbers will reduce in the future due to health campaigns
directed at making people more aware of the dangers of second hand smoking, and other
programs that have helped and are still helping in bringing down the rates of smoking .
Progress is also been made at genomic level .In august 2011 the FDA approved the drug
crizotinib(Xalkori) produced by Pfizer. This drug is a competitive inhibitor of ALK (anaplastic
lymphoma kinase). This drug is used for late stage lung cancer patients with a mutation in their
ALK gene. Studies show it gives these patients an additional 4.7 months when compared to
chemotherapy alone (MacConail 2012). Thus with continuous lung cancer research , funding
and sensitisation of the people on the dangers of smoking , rates of lung cancer could be
drastically reduced for though it is one of the deadliest cancers, it is also one of the most
Figure 1
Figure 1: the above graph shows cell growth inhibition by buffalin. One can see that as the dose
increases, the rate of inhibition increases on the y – axis and as the time the cells spend in the drug
increases the rate of inhibition also increases
Figure 2
Figure 2 : histograms from flow cytometry show the continuous decrease in the number of cancerous
cells as the dose and time in contact with buffalin increases. One can clearly see the high amounts of
apoptosis in sample 8 compared to little apoptosis in sample 2 because sample 8 was in contact with
buffalin for 72 hrs and had 100nm of buffalin while sample 2 was in contact for 48hrs with a 20nm
dose. Thus one can see the time and dose dependent apoptosis induced by buffalin
Figure 3
Figure 3 : a graph showing the rate of apoptosis when treated with buffalin and without treatment
(control). Here one can see that apoptosis is significantly low in the untreated control cells but
significantly high in the high dosed treated cells.
Figure 4
Figure 4: microscopic images of cells treated with buffalin(b) and control cells without treatment (a). in (b), one
can see the morphological changes of cytoplasmic shrinkage, membrane degradation and apoptotic bodies
associated with apoptosis. Proving that apoptosis is contributing to the low cell count of the tumours.
Figure 6
Figure 5
Figure 5: western blot results showing
Figure 6 : western blot results showing
The gradual increase in bax(pro apoptotic )
an increase in the amounts of active
and decrease in Bcl -2 (anti apoptotic) while
caspase 3 when cells are treated with
B- actin acts as control
bufalin. Thus increase in apoptosis
Figure 7
Figure 7: the graph shows the differences between cells treated with bufalin, those threated with bufalin and Z- DEVD –
FMK(caspase 3 inhibitor), and untreated cells (control). Here one can see the decrease in apoptotic rate when buffalin is used
with a caspase 3 inhibitor compared to when it is used alone. This decrease in apoptosis in the presence of a caspase -3
inhibitor signifies the regulatory effects of bufalin on caspase 3.
Figure 8
Figure 8: graphs showing flow cytometry results of the synergistic effects buffalin and LY294002 (Akt inhibitor).
Here one can see that buffalin alone induces apoptosis but when the cells are pre treated with an Akt inhibitor
there is a significant increase in the number death cancerous cells. This then indicates that Akt plays a big role in
the proliferation of cancerous cells and the inhibition of its pathway by buffalin is very essential in overcoming
chemo resistance.
Figure 9
Figure 5: western blot analysis showing the amounts of apoptotic proteins when bufalin works with LY294002 in
A 549 cells. Here one can see that buffalin works synergistically with LY294002(Akt inhibitor) to increase Bax
and , active caspase -3 . They also decrease pro caspase -3 , Bcl – 2 and phosphorylated Akt (pAKt) while the Akt
serves as a control. Thus bufalin induces apoptosis by inhibiting the Akt pathway, increasing pro apoptotic
proteins and decreasing anti apoptotic proteins.
Okur, Y., Yucel,B., Atasever,E., Eren, M.(2012) . Retrospectively evaluate prognosis and Treatment
results of patients with non small cell lung cancer. HealthMED, 6(5). Retrieved from (Academic search).
Zhu, Z., Sun,H., Ma, G., Wang,Z., Li, E., Liu, Y., Yunpeng, L. (2012) Bufalin Induces Lung Cancer Cell
Apoptosis via the Inhibition of PI3K/Akt Pathway. International Journal of Molecular Sciences, 13, 2025
– 2035. doi:10.3390/ijms13022025
MacConail,l.E. (2012) Advancing Personalized Cancer Medicine in Lung Cancer. Archives of
Pathology & Laboratory Medicine,136 1210 – 1216. doi: 10.5858/arpa.2012-0244-SA
Xie, C., Chan, Y.,W., YU,S., Zhao,J., Cheng, C.(2011). Bufalin induces autophagy-mediated cell
death in human colon cancer cells through reactive oxygen species generation and JNK
activation. Free Radical Biology and Medicine 51 (7) , 1365 – 1375. Retrieved from
(science direct)
Huang, W., Yang, J., Pai,S., Chang, S., …….. Chung,J.(2012). Bufalin induces G0/G1 phase arrest
through inhibiting the levels of cyclin D, cyclin E, CDK2 and CDK4, and triggers apoptosis via
mitochondrial signaling pathway in T24 human bladder cancer cells. Mutation
Research/Fundamental and Molecular Mechanisms of Mutagenesis, 732 (1- 2), 26-33. Retrieved
06(science direct)