Journal of Basic and Clinical Pharmacy

www.jbclinpharm.com

Effect of taxol from Pestalotiopsis mangiferae on

A549 cells-In vitro study
Govindarajan Kathiravan*1 and Sripathi M. Sureban 2,3

1) Department of Biotechnology, VELS University, Pallavaram, Chennai-600 117

2) Department of Medicine, Digestive Diseases and Nutrition Section, The University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma, USA 73104
3) Department of Veterans Affairs Medical Center, Oklahoma City, Oklahoma 73104

Abstract: Pestalotiopsis mangiferae Coelomycete fungi were used to examine the Keywords:
production of taxol. The taxol isolated from this fungus is biologically active against Pestalotiopsis mangiferae;
cancer cell lines were investigated for its antiproliferative activity in human Non Small taxol;
Cell Lung Cancer A549 cells. The results showed that the methylene chloride extrac- MTT; Trypan blue;
tion of Pestalotiopsis mangiferae inhibited the proliferation of A 549 cells as measured G0/G1; LDH;
by MTT and Trypan blue assay. Flow cytometric analysis showed that methylene chlo- apoptosis
ride extraction of Pestalotiopsis mangiferae blocked cell cycle progression in G0/G1
phase. In addition fungal taxol induced A549 cell apoptosis as determined by propid-
ium iodide staining. Further the percentage of LDH release was increased at increasing received on 03-01-2010
concentrations which is a measure of cell death. The levels of sialic acid levels and
modefied on 22-01-2010
DNA, RNA and protein levels were decreased after treatment with methylene chloride
extraction of Pestalotiopsis mangiferae.We suggests that methylene chloride extrac- accepted on 02-02-2010
tion of Pestalotiopsis mangiferae might be considered for future therapeutic applica- available online 15-02-2010
tion with further studies against lung cancer. www.jbclinpharm.com

Introduction of taxol against certain human cancer. Its mode of

L
ung cancer is the leading cause of cancer death action is unique in that it inhibits mitosis through
in the world and Non Small Cell Lung Carci- enhancement of polymerization of tublin and conse-
noma (NSCLC) accounts for approximately quent stabilization of microtubles during the process
75–85% of these cancers. Non small cell lung can- of cell division. However, a complete treatment for
cers commonly develop resistance to radiation and the patient requires 2 grams of taxol, administered
chemotherapy, and they often present at stages be- several times and many months. To obtain 1kg of
yond surgical remedy. Since current treatment mo- taxol requires about 10,000 kg of bark, and several
dalities are inadequate, novel therapies are necessary thousand tresses must be cut to procure this quan-
to reduce the effects of the increasing incidence in tity of bark. This scarcity of taxol and the ecologi-
pulmonary neoplasm (Chen et al., 1995; Park et al., cal impact of harvesting it encouraged scientists to
2001). find alternative methods using microorganisms. A
Taxol, a diterpene was originally isolated from hypomycetous fungus namely Taxomyces andreanae
the bark of Pacific Yew tree (Taxus brevifolia) more on Taxus sp., could produce taxol. A coelomycetous
then two decades ago and has proved to possess an fungus, pestalotiopsis microspora, an entophyte from
anticancer activity. The US National Cancer Insti- inner bark of Taxus wallachiana produced taxol in
tute, in collaboration with Bristol Myers Squibb Co culture. Keeping in this mind, an attempt has been
and other workers have demonstrated the efficacy made to examine the production of taxol by some
*Corresponding Author:
other coelomycetous fungus as well. The taxol isolat-
E-mail: gkathir72@gmail.com ed from these fungi is biologically active against can-

1
 Govindarajan Kathiravan et al. / Journal of Basic and Clinical Pharmacy

cer cell lines. In order to the price of taxol and make mg, KNO3-80 mg, KCl-60 mg, MgSO4-360 mg,
it more available, a fermentation process involv- NaH2PO4-20 mg, H3BO3-1.4 mg, MnSO4-5.0 mg,
ing a microorganisms would be the most desirable ZnSO4-2.5 mg and KI-0.7 mg.
means supply. It was first discovered by Strobel et al
(1997)., that the fungus Taxomyces andreanae could Extraction of taxol
produce taxol, through the yield was low. (Strobel et Extraction of taxol was performed according to
al., 1996) showed that Pestalotiopsis microspore iso- Strobel et al. (1996). After incubating the fungal
lated from the bark of Taxus wallachiana produced culture for 3-4 weeks, the culture filtrate was passed
taxol in mycelial culture. This work prompted us to through four layered cheesecloth. In order to avoid
continue the search for the taxol production from fatty acid contamination of taxol, 0.25g of NaCO3
fungal sources. was added to the filtrate. The culture fluid was ex-
Apoptosis has been characterized as a funda- tracted with two equal volumes of methylene chlo-
mental cellular activity to maintain the physiologi- ride and the organic phase was evaporated to dryness
cal balance of the organism. It is also involved in under reduced pressure at 35⁰C.
immune defense machinery (Hengartner, 2000)
and plays a necessary role as a protective mecha- 2.2. Cell culture
nism against carcinogenesis by eliminating dam- A549 was obtained from NCCS Pune, India. A 549
aged cells or abnormal excess cells proliferated ow- cells were cultured in Ham’s F12k medium contain-
ing to various chemical agents induction (Brown ing 10% new born calf serum containing 100µl/ml
and Wooters, 1999). Emerging evidence has dem- of penicillin and streptomycin 100µl/ml. Cells were
onstrated that the anti cancer activities of certain maintained in a humidified atmosphere of 5% Co2
chemotherapeutic agents are involved in induction incubator at 37⁰C, until confluency stage is attained.
of apoptosis, which is regarded as the preferred way The medium is replaced every two days and main-
to manage cancer. tenance is in strictly accordance with the standard
methods. The cells were dissociated with TPVG in
MATERIALS AND METHODS Phosphate buffered solution.
Source of chemicals
Fetal bovine serum (FBS), Penicillin G, Streptomy- Light microscopy
cin and amphotericin B were obtained from Hi Me- After 48 hours incubation with methylene chloride
dia. Dimethylsulfoxide (DMSO) and RPMI–1640 extract of fungal taxol, the A-549 cells were washed
were purchased form King Institute of Preventive with PBS.The cells were observed for morphological
Medicine. MTT and propidium iodide was pur- changes under light microscopy at 100X and photo-
chased from SRL, Laboroties India. All other chem- graphed.
icals used were of analytical grade.
Cell viability assay
Fungal material and extract preparation Inhibition of cell proliferation by methylene chlo-
The general laboratory techniques followed in the ride extract of fungal taxol, was measured by MTT
course of the present investigation were as outlined assay as described (Mossmann, 1983). Briefly, cells
by Booth (1971). The test fungi used in the present were plated in 24 well culture plates (1x106cells/
study were grown in Erlenmeyer flasks containing well). After 24 hrs incubation, cells were treat-
500 ml MID medium supplemented with 1 gram of ed with crude extract (10µgs, 25 and 50 µgs) for
soytone L-1 (Pinkerton and Strobel, 1976) for taxol 48 hrs. Fifty microlitres of MTT was added and
production. Three mycelial agar plugs (0.5 cm) were the reading was taken at 570nm after lysing in
used as inoculums. The organisms were grown at isopropanol.
24±2⁰C statistically for 3-4 weeks.
Trypan blue exclusion studies
Culture media (Strobel, 1996) The viability of cells were assessed by trypan blue
MID medium was supplemented with soytone, exclusion studies for various concentrations (10, 25
sucrose -30.0 g/l, (CHOH.COONH4) 2-5.0 g, and 50µgs/ml) at different time intervals 12, 24 and
Yeast extract -0.5g, Soytone-1.0g, Ca2(NO) 3-280 48 hrs by the standard method (Moldeus, 1978).

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 Govindarajan Kathiravan et al. / Journal of Basic and Clinical Pharmacy

Assay of lactate dehydrogenase (EC 1.1.1.27) iodide staining. For determination of apoptosis by
The activity of lactate dehydrogenase was assayed by propidium iodide staining, the A549 cells were treat-
the method described (King, 1965 a). ed with methylene chloride extract of fungal taxol
and stained with propidium iodide (50 µgs/ml). This
Assay of sialic acid levels is then viewed under the fluorescent microscope.
Sialic acid level were determined as according to the
method described(Warren, 1959). Flow cytometry
This analysis was carried according to the method
Estimation of macromolecules described by Nicotelli et al (1991). 10 lakh cells of
Protein was estimated by the method of Lowry et A549 were cultured in 6 well plates and incubated
al (1951). DNA was estimated as according to the for 48 hours to obtain a monolayer. To this culture,
method of Burton (1956). RNA was estimated by the drug was added at 50µgs concentrations and in-
the method of Rawal et al (1977). cubated for a period of 24 hours. The cellular mor-
phology was viewed once every few hours. After the
Measurement of apoptosis by propidium io- incubation period, the media was removed and the
dide staining cells gently washed with PBS. The cells were then
The induction of apoptosis by methylene chloride trypsinised. The suspension was then centrifuged at
extract of fungal taxol was assayed by the propidium 3000 rpm for 5 minutes and supernatant was dis-

Figure 1a-1f: showing the morphological features of A 549 cells treated with various concentrations of the ex-
tract (viz.control,10-50 micrograms).
1a 1d

1b 1e

1c 1f

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 Govindarajan Kathiravan et al. / Journal of Basic and Clinical Pharmacy

carded. The pellet obtained was gently washed with Architecture of untreated A549 cells show a typi-
ice cold PBS and resuspended. To this 40µl of 2mg/ cal spindle shaped cells observed under a light mi-
ml propidium iodide was added and kept for 4 hours croscope. It exhibited typical carcinoma type mor-
of incubation at 4oC. This was subjected to flow cy- phology with a uniform monolayer (figure 2.1). In
tometric analysis. vitro incubation of A-549 cells with the methylene
chloride extract of fungal taxol (1-5µgs) showed re-
RESULTS markable morphological alterations (figure 2.2-2.6)
TLC analysis was carried out on Merck 1mm(20x20 and cell death at the end of 48 hours characterized by
cm) silica gel plate developed in solvent A disruption of monolayer. It caused a dose dependent
(chloroform:methanol,7:1v/v) followed by sol- cytolytic and nuclear change in cell morphology. Cell
vent B (Chloroform: Acetonotrile, 7:3v/v), solvent mitosis were scarce compared to untreated A549
C (Ethyl acetate: 2-propanol, 95:5,v/v) solvent D cells.
(Methylene chloride: Tetrahydrofuran,6:2v/v) and Figure 3 showed the effect of methylene chloride
solvent E (Methylene chloride: Methanol:Dimethylf extract of fungal taxol on A549 cell viability (Trypan
ormamide,90:9:1v/v/v) respectively. The area of the blue uptake) after 12, 24, 48 hrs incubation. It was
plate containing putative taxol was carefully removed inferred that the incubation of methylene chloride
by scrapping off the silica at the appropriate Rf and extract of fungal taxol had led to marked decrease in
eluted with acetonitrile. Taxol was detected with 1% viable cells. The A549 cells after 12, 24 and 48 hours
w/v vanillin/sulphuric acid reagent after gentle heat- incubation to serial dilutions (10-50 µgs) showed de-
ing (Cardellina, 1991). It appeared as a bluish spot creased cell viability. The cell death was found to be
that faded to dark grey after 24 h. higher after 48 hours. Incubation of cells methylene
chloride extract of fungal taxol produced 48% cell vi-
Figure 2: showing the effect of fungal taxol of ability at 50 µgs after 24 hours and 10% cell viability
P.mangiferae on cell viability detected by trypan blue at 50 µgs after 48 hours (p>0.05). The experiments
assay.
were carried out in duplicate triplicates.
Cell viability was observed in a dose and time de-
pendent manner in taxol treated cells at increasing
concentrations ranging from 10,20,30,40 and 50µgs/
ml. At 24 and 48 hours, less than 50% of cell viability
was observed at a concentration of 30 micrograms/
ml. Maximum cytotoxic activity was observed at a
concentration of 50µgs/ml at 48 hours.
As evident from the figure 4, the cytotoxicity was
observed at increasing concentrations i.e.10, 20, 30,
The experiments were carried out in duplicate triplicates.All the values were statistically 40 and 50µgs (p<0.05). Even at 30 µgs, less than
significant at p<0.05.

Figure 3: showing the effect of fungal taxol of P. mangif- Figure 4: showing the effect of fungal taxol of P. mangif-
erae on cell viability detected by MTT assay. erae on cell viability detected by LDH assay.

48 hours 48 hours

The experiments were carried out in duplicate triplicates. All the values were statistically The experiments were carried out in duplicate triplicates. All the values were statistically
significant at p<0.05. significant at p<0.05

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 Govindarajan Kathiravan et al. / Journal of Basic and Clinical Pharmacy

Figure 5: shows the levels of sialic acid treated with 50% of cell viability is observed while at 50 µgs, only
fungal taxol of P. mangiferae. 10% of cell viability was seen.
A dose dependent increase in the percentage of
LDH leakage was observed. A maximum leakage of
LDH was observed at a concentration of 50µgs/ml
at 48 hours in taxol treated A549 cells. From the fig-
ure 5, the percentage of LDH release was increased
at increasing concentrations of methylene chloride
extract of fungal taxol which is a direct proportion to
cell death. At 50 µgs, the maximum of 90% cell death
was observed (p< 0.05) after 48 hours.
48 hours Fig 6 represents the levels of sialic acid in the
treated and untreated A-549 cells at a concentra-
The experiments were carried out in duplicate triplicates. All the values were statistically
significant at p<0.05. tion of 50µgs/ml. A marked reduction (p<0.05)
in the levels of sialic acid was observed in the taxol
Figure 6: showing the effect of fungal taxol of P. mangif-
erae on apoptosis detected by propidium iodide stain- treated A549 cells after 48 hours when compared to
ing. (6a-control, 6b- 40 µgs, 6c- 80µgs). untreated cells.
Figure 7 represents the fluorescent microscopic
6a
pictures of the methylene chloride extract of fungal
taxol treated and untreated A549 cells assessed for
apoptosis at 400X.The propidium iodide stained
cells at a concentration of 40 and 50 µgs/ml showed
the clumping of cells with slight distortion. The
highly condensed and fragmented nuclei that are the
index of apoptosis were observed at 40 and 50 µgs/
ml (fig 7b and 7c). The untreated A549 cells were
shown in plate 7a.
Table 1 shows the levels of DNA, RNA and pro-
6b
tein in taxol treated (50 µgs) A 549 and untreated
A549 cells. A significant decrease (p<0.05) in the
levels of these macromolecules was noted when com-
pared to untreated cells.
Plate 7.1 depicts the flow cytometric analysis of
untreated A 549 cells. Plate 7.2 depicts the drug
treated A 549 cells. Flow cytometry analysis for ap-
optosis study was carried out on the A549 cells after
the methylene chloride extract of fungal taxol treat-
ment. It was found that 56.06% of the cells were in
the G1 phase, 8.60% in the S phase and 35.78% of
cells in the G2 phase in the control cells of A 549
6c

TABLE 1: showing the effect of fungal taxol of P. mangif-

erae on DNA, RNA and protein levels.

Parameters Untreated Treated(50 μgs/ml)

DNA 10.86 ± 1.56 8.55 ± 1.71*

RNA 14.46 ± 1.75 11.88 ±1.06*

PROTEIN 967.07 ±32.86 917.14 ± 32.89*

The experiments were carried out in duplicate triplicates.All the values were statistically
significant at *p<0.05, NS-non significant when compared to untreated group (students
t-test).Values are expressed as μg/106 cells.

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 Govindarajan Kathiravan et al. / Journal of Basic and Clinical Pharmacy

Figure 7.1: shows the flow cytometric analysis of control cells and figure 7.2 shows the flow cytometric analysis
of fungal taxol of P. mangiferae treated A 549 cells.

cells. The methylene chloride extract of fungal taxol the methylene chloride extract of fungal taxol. MTT
(5 µgs) treated cells had only 28.64% of the cells in is cleaved by tetrazolium ring by succinate tetrazo-
the G1 phase, 4.4% in the S phase and the remaining lium reductase in active mitochondria. Metabolically
50.46% cells in the G2 phase. active cells cleave MTT and generate a formazan
product, which forms purple crystals, and colour
Discussion developed is directly proportional to cell number
A number of studies have indicated that significant (Mossman, 1983).
cell proliferation effect against various cancer lines. Based on the results of the cell viability and % of
In our work, we demonstrated that the methylene LDH leakage, a concentration of 5µg/ml for further
chloride extract of fungal taxol extract inhibited the biochemical studies (results not mentioned). LDH
cell proliferation in A549 by inducing apoptotic cell leakage is routinely used as an indicator of damage
death. The cytotoxic effect was clearly established at to plasma membrane integrity and in assessing cyto-
increasing concentrations i.e.1, 2, 3, 4 and 5 micro- toxic nature of the plant, as dead cells release LDH
grams. The cytotoxic effect was characterized by dis- in to the culture medium (Matsuda et al., 1980). In
ruption of the monolayer. (Kathiravan and Muthu- the present study at 48 hours of incubation a maxi-
mary 2009) mum leakage of LDH leakage was observed at a
Cancer chemotherapeutic as well as chemopre- concentration of 5µg/ml, which might be due to the
ventive agents exert part of their pharmacological cytotoxic effect of taxol leading to the loss of plasma
effect by triggering apoptotic cell death or cell cycle membrane integrity. Based on the results of cell vi-
transition. Identification of inhibition of apoptosis ability and % LDH release, a concentration of 5µgs/
by several tumor promoters and induction of apop- ml was taken for further studies.
tosis in tumor cells, serves as a predictor of tumor An important component of the cellular response
treatment (Kim et al., 1999). Extracts from a broad to DNA damage is the inhibition of DNA synthesis.
spectrum of plant species contain substances that DNA damage could be achieved through in inhibi-
possess antitumor activity (Dzham et al., 2002). tion of a positive regulatory pathway of DNA syn-
The MTT assay, which measures the formazan thesis and or costimulaton of a negative regulatory
product at 570 nm, clearly proves the cytotoxicity of pathway. The ability to inhibit DNA synthesis arises

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 Govindarajan Kathiravan et al. / Journal of Basic and Clinical Pharmacy

Figure 7.1: shows the flow cytometric analysis of control cells and figure 7.2 shows the flow cytometric analysis
of fungal taxol of P. mangiferae treated A 549 cells.

from the possibility of interface with DNA replica- The PI stained cells at a concentration of 5 µgs/
tion enzymes and possibility to induce apoptosis ml showing clumping of cells with slight distortion.
(Kajimoto et al., 2002). A growth inhibitory and apoptosis inducing activity
Sialic acid is widely distributed in mammals and of many flavanoids with the release of cytochrome c
usually occurs as a terminal component at the non is already established (Bauer et al., 1997; May and
reducing end of carbohydrate side chains of glyco- May, 1999). Evidence indicates that flavanoids may
proteins and glycolipids. A increased concentration promote apoptotic cell death. The results of the
of sialic acid is observed in the cell surface and sialo present study reveal that taxol promotes apoptotic
groups are separated by some of these cells (Gorc- mode of cell death in A549 cells which may be attrib-
zyca et al., 1993). Elevation of sialic acid levels is uted to the presence of flavanoids. Detailed investi-
commonly found in cancer cells (Kastan et al., 1992). gations are required to establish the actual flavanoids
Similar results have been observed in the present involved and possible potential of taxol in cancer
study with A 549 cells. A decrease in sialic acid levels control and chemotherapy.
noticed in taxol treated A549 cells of present study. In the cells undergoing apoptosis, DNA was de-
This could be attributed to the presence of flavanoids graded to fragments of low molecular weight and
in the taxol extract which could mediate the mem- subsequently leaked out from the cells and the DNA
brane permeabilisation and bring about modifica- content was stained with a DNA-specific flouro-
tions in the glycoprotein’s components as secondary chrome, propidium iodide (PI), a special DNA peak
biochemical responses. (usually called sub-G1 peak) appeared. The G0/G1

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 Govindarajan Kathiravan et al. / Journal of Basic and Clinical Pharmacy

population in the methylene chloride extract of fun- of S phase cells. Thus the blockage effect of methyl-
gal taxol treated A 549 cells was increased after 24 ene chloride extract of fungal taxol occurred at G1/S
hours at 5 µgs/ml. Our cell cycle analysis by flow cy- transitions and thus increase of cell numbers in G1
tometry showed that there was a prominent increase phase was clearly due to decrease of cells in S phase.
in the G0/G1 DNA upon methylene chloride extract Much research has showed that the arrest of the cells
of fungal taxol treatment. This increase in the G0/ at the checkpoints of the cell cycle occurs as an event
G1 DNA is an indication of the inhibition of DNA preceding the detection of apoptotic cells (DeAlbu-
replication. The increase of DNA content indicated ruerque et al., 2004).
the retardation of cell cycle, which might have taken There may be some factors that affect the relation
place during the G1-S transition phase. The possible of test compound effect between in vivo and in vitro,
mechanism of action would be down regulation of including culture conditions, tumor microenvironment,
the activity of cyclin E dependent kinase, which plays drug distribution, active metabolites and indirect inhi-
an essential role for cell cycle progression at the G1/S bition effects on tumor proliferation (e.g. angiogenesis).
transition stage. This inhibition of cell-cycle progres- Rather cell culture should be used to illustrate principles,
sion might be associated with an altered expression concepts and mechanisms of action that may be active in
of cell cycle relevant regulator, including p21 and its vivo. If we see it from a drug development point, there
upstream molecule p53 (Kuo, 1996). are numerous efforts being made to find a method of de-
Cell cycle control has been proven to be a major livery to achieve the concentration of crude extract in cell
event in ensuring the accurate cell division. Abnor- cultures.
malities of cell cycle regulators have been associated The idea of study is that to show efficacy in cell
with many carcinogenic processes. The data suggests cultures first, then start evaluating effects in animals.
that methylene chloride extract of fungal taxol could Taxol from Pestalotiopsis mangiferae appears to be
cause a significant accumulation of cells in G0/G1 a promising drug for its future use as an anticancer
phase in human lung adenocarcinoma cells (A549) drug. From our observation, we do agree it is only a
after 24 hours. Our data clearly showed that increase preliminary study and it requires a further detailed
of G0/G1 phase cells was accompanied by decrease study.

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