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Mycology
An International Journal on Fungal Biology
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Modulation of Bax and Bcl-2 genes by secondary
metabolites produced by Penicillium rubens JGIPR9
causes the apoptosis of cancer cell lines
Prerana Venkatachalam & Varalakshmi Kilingar Nadumane
To cite this article: Prerana Venkatachalam & Varalakshmi Kilingar Nadumane (2019): Modulation
of Bax and Bcl-2 genes by secondary metabolites produced by Penicillium�rubens JGIPR9 causes
the apoptosis of cancer cell lines, Mycology, DOI: 10.1080/21501203.2019.1707315
To link to this article: https://doi.org/10.1080/21501203.2019.1707315
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
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Published online: 26 Dec 2019.
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Modulation of Bax and Bcl-2 genes by secondary metabolites produced by
Penicillium rubens JGIPR9 causes the apoptosis of cancer cell lines
Prerana Venkatachalam and Varalakshmi Kilingar Nadumane
Department of Biotechnology, School of Sciences, JAIN (Deemed-to-be University), Bengaluru, India
ABSTRACT
Search for an efficient anti-cancer compound of natural origin with well-defined mechanisms of
action is an important scientific pursuit today, due to cancer being the second leading cause for the
death of affected people. The members of the genus Penicillium are one of the important sources
of bioactive compounds. In the present study, Penicillium rubens, isolated from a garden soil in
Madurai district of Tamil Nadu, was found to produce a highly promising anti-cancer metabolite.
The percentage viabilities of HepG2, HeLa and MCF-7 cancer cells treated with the bioactive
fraction (P5) isolated from P. rubens, ranged between 40-50% after 96 h. Apoptosis induction
was found to be the major reason for the observed reduction in cancer cell proliferation and cell
count which was confirmed by caspase activity, DNA fragmentation, clonogenic assay, cell cycle
analysis and LDH assays. The upregulation of proapoptotic Bax, coupled with the downregulation
of anti-apoptotic Bcl-2 expressions were confirmed by RT-qPCR and flow cytometry methods. The
current study also indicated an upregulation of p53 which further strengthened the apoptogenic
property of P5 fraction. Non-toxicity of P5 was demonstrated on normal peripheral lymphocytes.
The analysis of P5 fraction through GC-MS indicated the presence of indole-2, 3-(4,4-dimethyl-
3-thiosemicarbazone) as one of the major compounds.
ARTICLE HISTORY
Received 29 September 2019
Accepted 3 December 2019
KEYWORDS
Cytotoxicity; caspase
activities; cell cycle; Bax
expression; secondary
metabolites; Penicillium
rubens
Introduction
Cancer is a dreadful disease affecting a large percen-
tage of humans and despite advanced treatment
modalities with the death rate being very high, the
goal for a successful cancer therapy demands the
identification of highly efficient therapeutic mole-
cules. The high toxicity and side effects associated
with anti-cancer drugs increases the need for novel
drugs active against diverse kinds of tumours, with
lesser or no side effects (Demain and Sanchez 2009).
Nature is considered to be one of the most important
sources for pharmacologically active compounds as
part of drug discovery (da Rocha et al. 2001;
Bhatnagar and Kim 2010). Natural products have
played a significant role in the treatment of cancer
over the past few decades.
Filamentous fungi have gained increased attention
and are being exploited extensively as they are the
producers of novel secondary metabolites of thera-
peutic significance which have proven as superior to
products of chemical origin and have shown their
usefulness as anti-cancer agents (Calvo et al. 2002;
Kinghorn et al. 2016). Penicillium sp. are a class of
fungi that are widely distributed in nature producing
secondary metabolites of pharmaceutical importance
such as the β-lactum antibiotics penicillin, xanthocil-
lins, sorbicillin, chrysogine among many others (De
Hoog et al. 2000; Mohammad et al. 2011). The mem-
bers of the fungal genus Penicillium have been utilised
worldwide for the production of highly versatile cyto-
toxic secondary metabolites. Many of the metabolites
isolated from various species of the genus Penicillium
have shown promising growth-inhibitory properties
against different in-vitro as well as in-vivo human
cancers (Kornienko et al. 2015; Koul and Singh 2017;
Youssef and Alahdal 2018). Several lead compounds
from terrestrial fungi are currently being used for
medicinal purposes (Gomes et al. 2015). It has been
estimated that only around 100,000 species out of the
approximately 3 million fungal species on Earth have
been described so far. Therefore, to find an efficient
anti-cancer compound from fungal source, which is
also safe to the normal healthy cells, filamentous
fungi were isolated from different sources and
screened for their cytotoxicity to in-vitro cancer cell
CONTACT Varalakshmi Kilingar Nadumane kn.varalakshmi@jainuniversity.ac.in
Supplementary data for this article can be accessed here.
MYCOLOGY
https://doi.org/10.1080/21501203.2019.1707315
© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Published online 26 Dec 2019
lines. Based on the results of initial screening, among
the isolates, Penicillium rubens JGIPR9 was chosen for
the current study.
Methods
Isolation of fungi
Filamentous fungi were isolated from many environ-
mental sources (air, water, soil and phylloplane) by
serial dilution method (Aneja 2003). The organisms
were isolated on M9 medium, supplemented with
1% casein. Different coloured fungi were selected
and screened for their cytotoxicity to cancer cell
lines. The fungal cultures were maintained in Czepak
dox yeast agar. The isolate with the highest cytotoxic
effect was chosen and identified by molecular
methods.
Extraction of secondary metabolites
A primary inoculum containing 2×10
6
fungal spores/ml
was used for the culture studies. The chosen fungus
was incubated in Czepak Dox Yeast Broth at 24-28°C
under static conditions for 12 days. The mycelium was
removed by filtering through Whatmann filter paper
and was dried overnight at 60°C. The dried biomass
was later homogenised and the metabolites were
extracted using methanol. The extract was evaporated
to dryness and the dried extract was used for initial
cytotoxicity screening.
Cell lines
Cancer cell lines MCF-7, HeLa and HepG2 were
acquired from NCCS, Pune, India. Healthy human per-
ipheral lymphocytes were used as control cells. Cells
were maintained in MEM (HiMedia) supplemented
with 10% FBS (HiMedia) and incubated at 37°C with
5% CO
2
.
Isolation of lymphocytes
Isolation of lymphocytes from blood was performed
as per the ethical guidelines outlaid by Indian Council
of Medical Research (ICMR) (Mathur 2017) following
a standard protocol (HiSep LSM 1077, HiMedia) with
the addition of phytohaemagglutinin-L (Himedia) to
stimulate proliferation.
MTT assay
Cells were seeded at an initial concentration of 1×10
4
cells/ml onto 96-well microtiter plates. After 24 h, the
methanol extract from the fungal biomass was added
at varying concentrations (1, 10, 50 and 100 µg/ml) for
24, 48, 72 and 96 h. The cytotoxic effect was assessed
by the addition of MTT dye as per the standard pro-
tocol (Mosmann 1983).
Fractionation by preparative thin layer
chromatography (TLC)
The methanol extract was partially purified by pre-
parative TLC using Silica gel 60 F
254
(Merck) as pre-
viously reported (Rabel and Sherma 2017). Each of the
partially purified fraction was screened for its cyto-
toxicity on cancer cells and the one with highest
activity was chosen for further studies.
Morphological observation
Morphological changes after 48 h of cancer cells (2x10
6
cells/ml) treated with sample (25 μg/ml) along with the
untreated control were observed under an inverted
microscope (Labomed, Germany) (Freshney 2000).
Fluorescence microscopy
Cancer cells (2x10
6
cells/ml) and healthy lymphocytes
were treated with the sample (25 μg/ml) for 48
h along with untreated control. The cells were stained
with acridine orange/ethidium bromide (AO/EtBr) and
observed under the fluorescence microscope
(Brousseau et al. 1999).
DNA fragmentation analysis
Cancer cells (2x10
6
cells/ml) were treated with the
sample (25 μg/ml) for 48 h along with untreated
control. The DNA from untreated and treated cancer
cells were isolated as per the previous methodology
(Gavrieli et al. 1992) and were electrophoresed on 1%
agarose gel along with 1kb ladder DNA (Genei).
Lactate dehydrogenase (LDH) activity assay
The LDH activity of the cancer cells (2x10
6
cells/ml)
treated with the sample (25 μg/ml) for 48 h was
2P. VENKATACHALAM AND V. K. NADUMANE
determined as per the kit manufacturer’s instructions
(G-Biosciences, cat. # 786–210) (F K-M et al. 2013).
Caspase −3, −7and −10 activities
Cancer cells (2x10
6
cells/ml) were treated with the
sample (25 μg/ml) for 48 h along with untreated
control. The activities of caspase −3, −7 and −10
were analysed as per the kit manufacturer’s instruc-
tions (G-Biosciences, cat. # 786–202 B) (Béchohra et al.
2016).
Nitric oxide (NO) assay
The NO release in cancer cells and healthy lympho-
cytes (2x10
6
cells/ml) treated with the sample (25 μg/
ml) for 48 h was estimated using standard protocol
(Ding et al. 1998). The nitrite concentration was
expressed as µM/ml.
Clonogenic assay
The ability of cancer cells to form colonies after sam-
ple treatment for 48 h at 25 and 50 µg/ml concentra-
tions were assessed by performing clonogenic assay
as per the standard protocol (Franken et al. 2006). The
cells were harvested by trypsinisation, washed with
PBS and immediately seeded onto culture flasks at
very low concentrations (100–200 cells/ml). The col-
ony formation was observed after a period of 14 days.
The colonies formed were washed with phosphate
buffer, fixed in acetic acid and methanol fixative for
30 min followed by staining with crystal violet (1%).
The number of colonies was determined by counting
the colonies with >50 cells as the minimum size for
a colony.
Cell cycle analysis
Stages of cell cycle were analysed for DNA content
using a BD FACS Verse flow cytometer (IISc,
Bengaluru) after treating the cancer cells (2x10
6
cells/ml) with the sample (25 μg/ml) for 48 h (UC
San Diego Health Sciences Protocol –https://med
school.ucsd.edu/research/moores/shared-resources
/flow-cytometry/protocols/Pages/cell-cycle-with-pi.
aspx). The percentage of cell populations in sub-G1,
G1, S and G2 phases was estimated using FACSDiva
version 6.1.3.
Analysis of Bcl-2, Bax and p53 expressions at the
mRNA and protein levels
The anti-apoptotic Bcl-2, pro-apoptotic Bax and
tumour suppressor p53 expressions at the mRNA
level were assessed by RT-qPCR and at the protein
level by flow cytometry as per the previous metho-
dology (Venkatachalam and Nadumane 2018).
Gas chromatography –mass spectrometry (GC-MS)
analysis
The GC-MS analysis was carried out for the fraction
P5 at The South Indian Textile Research Association
(SITRA), Coimbatore. The analysis was performed
with a Thermo GC-Trace Ultra Ver: 5.0, Thermo
MS DSQ II in DB 35 –MS Capillary standard non-
polar column, dimensions: 30 m (L) x 0.25 mm (ID)
x0.25µmfilm thickness. The carrier gas used was
Helium and the flowratewas1ml/min.1µlofthe
sample was injected to the column. The initial
temperature for the program was 70°C raised to
260°C at 6°C/min, total time was 37.50 min. Scan
mass range was 50–650 m/z. The spectrum of the
unknown compound was analysed using an online
database for anti-cancer molecules (Cancer
Resource Database –http://data-analysis.charite.
de/care/).
Statistical analysis
All of the experiments were performed thrice and the
results were expressed as mean ± standard error.
Significance of the results were checked by one-way
/two-way ANOVA (GraphPad Prism 6.0 software).
Ap-value corresponding to <0.05 was taken for
reporting statistical significance.
Results
Identification of the organism
Out of the 35 fungal isolates, the one which was
isolated from garden soil obtained from Madurai dis-
trict, Tamil Nadu was exhibiting promising cytotoxic
and anti-cancer properties. The isolate was identified
as Penicillium rubens JGIPR9 (Accession no.
MH816935) by performing 18 s rRNA sequencing
(Biokart India Pvt. Ltd., Bengaluru).
MYCOLOGY 3
Evaluation of cytotoxicity
When different concentrations of the methanol
extract from the fungal isolate were checked for
its cytotoxicity against cancer cells, significant cyto-
toxic effect was observed (Figure 1(a)). The highest
cytotoxic effect was on MCF-7 cells at 100 µg/ml
concentration after 72 h of treatment with
a viability of 39.09%, followed by HepG2 cells
with 40.54% and HeLa cells with 60.34%, both
after 96 h treatment.
When the methanol extract was subjected to prepara-
tive TLC for fractionation, ethyl acetate and acetone (1:1)
used as the solvent system resulted in separating five
fractions (Bands P1 –P5). The cytotoxic effect of all the
fractions were tested against the cancer cell lines and
band 5 (P5) was found as the one with highest effects
(results not shown). This fraction was checked for its anti-
proliferative effects at 1, 10 and 25 µg/ml concentrations.
The cytotoxic effect was significant at 25 µg/ml concen-
tration with a viability of 49.74% in HeLa cells after 48 h,
42.92% in HepG2 cells after 96 h and 40.49% in MCF-7
cells after 96 h (Figure 1(b)). The IC
50
values were calcu-
lated as 24.4 µg/ml for HepG2, 11.0 µg/ml for HeLa and
16.0 µg/ml for MCF-7 cells. The viability of lymphocytes
was found to be higher than 98% at all the tested con-
centrations even after 96 h of incubation period with
both crude and TLC purified fractions (Figure 1(a,b)).
When the cytotoxic effect of tamoxifen citrate,
the positive control, was tested on cancer cell
lines and lymphocytes, there was a significant
cytotoxic effect with 62.1% viability in MCF-7
cells, followed by 67.2% and 72.14% in HepG2
and HeLa cells, respectively, after 72 h at 25 µg/
ml treatment concentration. The viability further
decreased to 56.5% in MCF-7 cells, 61.8% in
HepG2 and 70.0% in HeLa cells after 96 h at the
same treatment concentration. In the case of
healthy lymphocytes, it had not demonstrated
any cytotoxic effects when treated at the same
concentrations (Figure 1(c)).
Figure 1. Anti-cancer effect of the methanol extract and P5 fraction from Penicillium rubensalong with tamoxifen citrate as positive
control. *p< 0.05 and **p< 0.01 indicate the levels of significance in comparison to the control.
4P. VENKATACHALAM AND V. K. NADUMANE
Morphological observation
After 48 h of P5 treatment to the cancer cell lines,
many morphological changes such as cell rounding
up, detachment from the substrate, cell shrinkage,
presence of apoptotic bodies along with a decrease
in cell concentration were clearly observed in HepG2,
HeLa and MCF-7 cells (Figure S1).
Fluorescence microscopy
After 48 h of P5 treatment, cancer cells were har-
vested, stained with AO/EtBr. Under the fluorescence
microscope, cells fluorescing green (Figure 2a(a–d))
represented the healthy living cells. Cells that fluor-
esced light orange to deep red (Figure 2a(e–g)) repre-
sented apoptotic cells. Figure 2(h) represents the
treated lymphocytes, which clearly were fluorescing
green, an indication of their viability, just like the
untreated lymphocytes. The safety aspect of P5 to
normal cells is evident here.
DNA fragmentation analysis
When the pattern of DNA fragmentation of cancer cell
lines treated with P5 for 48 h were analysed, it appeared
as a smear in all three cancer cell lines as compared to
the intact band in case of untreated control cells
(Figure 2b(i–k)).
LDH activity
When the LDH activity assay was performed after 48
hof P5 treatment (Figure 3(a)), the highest cytotoxicity
Figure 2. (a). Fluorescence microscopic photographs after AO/EtBr staining. Arrows indicate apoptotic bodies/chromatin condensa-
tion. C –control and T –P5 treated; Scale bar: 20 μm. (b): DNA fragmentation analysis of HeLa, HepG2 and MCF-7 cells.
MYCOLOGY 5
was observed against HeLa cells (60.37%) followed by
MCF-7 cells with 57.38% and HepG2 cells with 43.29%.
A negligible cytotoxicity (2.9%) was observed on the
lymphocytes due to P5 treatment.
Caspase activities
The activities of caspase −3, −7and −10 were 20.72%
in P5 treated HeLa cells as compared to 16.06% in its
respective untreated control. A higher activity of
56.84% was observed in P5 treated MCF-7 cells
when compared to the 23.15% in the untreated con-
trol. The highest caspase activity was observed in
HepG2 cells with 144.54% activity after P5 treatment
when compared to the 23.68% in the untreated
HepG2 cells (Figure 3(b)).
NO assay
When culture supernatants of the treated cells were
evaluated for their NO levels, an increase in NO was
observed. The NO release was estimated to be 15.75
µM, 15.33 µM and 16.48 µM as compared to 11.62 µM,
11.58 µM and 13.23 µM of NO in untreated HepG2,
HeLa and MCF-7 cell lines, respectively, however, the
NO level in lymphocytes after treatment was found to
be unaltered (Figure 3(c)).
Clonogenic assay
Through clonogenic assay, fewer number of colonies of
the cancer cells were formed after treatment, which
were comparatively smaller in size than the untreated
cells (Figure 3(d)). There was a clear reduction in the
number of cells present in a single colony and the
number of colonies formed due to P5 treatment at 25
and 50 µg/ml concentration. The colony formation
potential was dose-dependent. The number of colonies
varied between 96-106 at 25 µg/ml concentration and
62–72 at 50 µg/ml concentration of P5, as compared to
the 130–190 colonies in the untreated control cells.
Stages of cell cycle
When the cell cycle of the P5 treated cells was checked
through flow cytometry, an increase from 4.86% to
Figure 3. Assays for LDH, Caspase, Nitric oxide and clonogenic ability.*p< 0.05 and **p< 0.01 indicate the levels of significance in
comparison to the control.
6P. VENKATACHALAM AND V. K. NADUMANE
62.65% in the sub-G1 phase and decrease from 59.77%
to 28.75% in G0/G1 phase together with a decrease in
S and G2/M phase was observed in HeLa cells (Figure 4(a)
and b). In HepG2 cells, major accumulation of cells was
observed in sub-G1 phase with 87.42% after treatment
against 16.39% of cells in controls. The population of
cells in G0/G1 phase was found decreased from 69.43%
to 5.72% together with a reduction in S and G2/M phase
cells (Figure 4(c,d)). A massive increase in the sub-G1 cells
and a decrease in G0/G1, S and G2/M phases of HeLa and
HepG2 cells clearly indicated apoptosis of the cells.
A similar trend of increased sub-G1 population from
4.66% to 46.24% after treatment was found even in
MCF-7 cells along with a decrease in G0/G1 phase with
13.38% cells as opposed to 69.5% cells in the controls.
There was accumulation of 29.41% cells in the G2/M
phase, suggesting a G2/M phase arrest (Figure 4(e,f)).
Contrary to these results, treatment of P5 to normal
lymphocytes resulted in an increased cell concentration
together with 20.38% sub-G1 cells in comparison to the
24.33% control cells (Figure 4(g,h)).
Bcl-2, Bax and p53 mRNA levels
The mRNA levels of Bcl-2, Bax and p53were analysed
in all three cancer cell lines (Figure 5a(a–c)). The
Figure 4. Flow cytometric analysis of cell cycle events after P5 treatment.
MYCOLOGY 7
concentration of total RNA isolated from both treated
and untreated cell lines ranged from 31.8 to 201.6 ng/
μl. After cDNA synthesis with specific primers, the
expression levels of p53, Bax and Bcl-2 were deter-
mined by RT-qPCR. The relative expression of Bax was
increased 47-folds in HeLa, 50.7-folds in HepG2 and
55.5-folds in MCF-7 cell lines after P5 treatment.
Simultaneous to this, the expression level of p53 was
also higher with a 68.1-folds in HeLa, 25.7-folds in
HepG2 and 82.3-folds in MCF-7 cell lines in compar-
ison to their controls. In the same group of treated
cells, the expression of Bcl-2 was highly downregu-
lated in HeLa cells corresponding to 14.3-folds
decrease followed by 2.6-folds decrease in MCF-7
and 1.3-folds decrease in HepG2 cells as compared
to their respective control cells.
Bcl-2, Bax and p53 protein levels
When the levels of the proteins Bcl-2, Bax and
p53were evaluated in the treated cancer cells through
flow cytometry (Figure 5b(d–f)), results similar to that
observed in RT-qPCR analysis were seen. It was found
that the Bcl-2 level was drastically decreased, i.e., from
99.8% to 9.19% in HeLa cells, from 99.35% to 0.77% in
MCF-7 cells and from 97.3% to 27.7% in the case of
HepG2 cells (Table S1). The findings are significant as
it is obvious that the regular cell cycle regulatory
mechanism is disrupted in cancer cells with high
expression of anti-apoptotic genes, thereby evading
death due to apoptosis. It was also seen that Bax
expression was upregulated, with an increase from
0.86% of the controls to 73.5% of treated HeLa cells
expressing it. The expression of Bax in HepG2 cells
increased from 0.33% in control cells to 48.6% and in
MCF-7 cells, it went up from 0.03% to 48.72% after 48
h of P5 treatment (Table S2). Along with these two,
the expression of p53 was also greatly enhanced in
the case of HepG2 cells with an increase from 1.25%
to 93.16%, in HeLa from 0.14% to 16.4% and in MCF-7
cells from 0.36% to 17.5% (Table S3).
Characterisation of P5 through GC-MS analysis
The GC-MS analysis of P5 resulted in five major peaks at
6.38, 25.03, 32.37, 35.98 and 37.77-min retention time
(Figure 6(a)). A thorough library search from the cancer
resource database (http://data-analysis.charite.de/care/)
indicated compounds such as1,2,5,6-Tetrathiocine with
an m/z ratio of 180.3 and Indole-2, 3-(4,4-dimethyl-
3-thiosemicarbazone), an indole-thiosemicarbazone
derivative with an m/z ratio of 248.3 (Figure 6(b)) from
Figure 5. (a). Expression patterns of the p53, Bax and Bcl-2 mRNA in response to P5 treatment. (b). Expression patterns of the p53, Bax
and Bcl-2 proteins in response to P5 treatment. C-control and T- P5 treated.
8P. VENKATACHALAM AND V. K. NADUMANE
the fraction at the retention time 35.98 min. Among
these, Indole-2, 3-(4,4-dimethyl-3-thiosemicarbazone)
has been reported to have anti-cancer activity. It has
been isolated earlier from plant sources and also from
Bufo frogs but not from Penicillium rubens (Ibrahim and
Elsaman 2018).
Discussion
Cancer is a disease that efficiently overcomes all kinds
of available therapies and the side effects of the exist-
ing therapeutic options are far more harmful than the
disease itself. Finding an alternative therapy is of
utmost importance in cancer management.
Filamentous fungi, especially of the genus
Penicillium, have contributed immensely towards
research in industrial and pharmaceutical domains
with several lead compounds towards cancer therapy
being reported from various species of Penicillium. But
till now no anti-cancer drug from Penicillium sp. has
been approved by FDA and reached the consumer
market. In the present study, a newly isolated strain of
P. rubens had demonstrated promising anti-cancer
properties to in-vitro cancer cell lines. Though there
are reports of secondary metabolites with antitumor
activity from Penicillium sp. (Bladt et al. 2013; Chavez
et al. 2015; Nicoletti and Trincone 2016; Feng et al.
2018; Farha and Hatha 2019; Orfali and Perveen 2019),
a metabolite from P. rubens having promising anti-
cancer properties has been reported for the first time
in this present work.
In the current study, the bioactive fraction (P5)
obtained from the partial purification of the methanol
extract from P. rubens JGIPR9 was tested for its anti-
cancer activity on HepG2, HeLa and MCF-7 cell lines.
The IC
50
value of the fraction was 24.4 µg/ml for
HepG2, 11.0 µg/ml for HeLa and 16.0 µg/ml for MCF-
7 cells. These IC
50
values are well within the range
suggested by the FDA. These values are better than
that reported earlier about the IC
50
value of fudeca-
dione A, isolated from Penicillium sp. BCC 17,468
(Pittayakhajonwut et al. 2011). The cytotoxic effect
of P5 on cancer cells was found to be significantly
higher than the positive control (tamoxifen) used in
the study. The cytotoxic effect of P5 was first con-
firmed by observations under both inverted and fluor-
escence microscopes, which revealed the presence of
fragmented particles, apoptotic bodies and reduced
cell numbers which are considered as hallmarks of
apoptosis.
Evidence for the direct cytotoxicity of P5 was pro-
vided by the results of LDH cytotoxicity assay. Loss of
Figure 6. GC-MS analysis of P5 fraction.
MYCOLOGY 9
membrane integrity due to the cytotoxic effects by
external agents causes LDH to release to the cyto-
plasm. Here, in the present study, after P5 treatment,
the percentage cytotoxicity was high in HeLa, HepG2
and MCF-7 cells which might be one of the triggering
factors that led to cell death observed in this case.
To be an efficient anti-cancer agent, a compound
needs to be having anti-proliferative effects too on
the cancer cells apart from exerting direct cytotoxic
effects. Proof for anti-proliferative property of P5 was
given by the results of clonogenic assay, which
resulted in a reduction (dose-dependent) in the num-
ber of colonies formed due to its treatment to the
cancer cells.
One of the major characteristics for an ideal drug
for cancer therapy is its efficiency to inhibit cell pro-
liferation and induce apoptosis in cancer cells. The
ability of P5 to induce apoptosis in the treated cells
was checked by analysing the DNA fragmentation
pattern, as it is a clear indicator of apoptosis happen-
ing in the cells. In the present study, the DNA of
treated cancer cells appeared like a smear upon elec-
trophoresis, indicating higher degree of apoptosis in
these cells (Paul et al. 2010; Mathi et al. 2014).
During DNA fragmentation, elevation of caspase
enzymes in cells is evident, as caspases play crucial
roles in the apoptotic machinery. The compound P5
in the present study was inducing higher caspase
activities in the cancer cell lines, thereby providing
proof for its apoptogenic property.
Bcl family of proteins are important regulators of
apoptosis, where Bcl-2 and Bax are the proteins
responsible for inhibiting and promoting apoptosis,
respectively. In addition to this, the tumour suppres-
sor p53 plays a significant role in controlling the cell
cycle progression along with inducing apoptosis
(Basu and Haldar 1998). In our study, as per the results
of RT-qPCR analysis, the mRNA levels of Bax and p53
were markedly increased, concomitant to a decrease
in Bcl-2 in the P5-treated cancer cells, but their levels
of expression varied slightly among these cells, which
might be due to the cell line specificity of P5. Flow
cytometry results indicated a similar trend of Bax, p53
protein upregulation and Bcl-2 protein suppression.
When these results were put together, we could
observe that the co-ordinated effects of the upregu-
lated Bax and p53 might be the driving force behind
the observed apoptosis in the cancer cells. Though
slight discrepancy was seen in the quantities of the
mRNAs and proteins, we can say that this could be
due to the differences in the regulation of transcrip-
tion and translation after P5 treatment (Maier et al.
2009; de Sousa et al. 2009; Liu et al. 2016).
Cell cycle analysis indicated an activation-induced
cell death or direct apoptosis is suggestive for HeLa
and HepG2 cells treated with P5 fraction, as an
increase in the sub-G1 phase cells was observed. In
P5 treated MCF-7 cells, G2/M phase arrest was
noticed. A similar trend of increased apoptotic cells,
i.e., sub-G1 phase was earlier reported due to the
addition of bioactive compounds on cancer cell lines
(Kajstura et al. 2007; Belayachi et al. 2017). A G2/M
phase arrest in combination with an increase in the
sub-G1 population has been reported earlier (Burgess
et al. 2006; da Silva et al. 2015). Though P5 treatment
resulted in increased sub-G1 phase and decreased cell
count in cancer cells, the same did not cause any
significant changes to the cell cycle of normal lym-
phocytes, clearly reflecting the specificity of P5 to the
cancer cells.
Another indicator of apoptosis is the NO release
from cells upon drug treatment. NO is synthesised by
three distinguishable forms of nitric oxide synthase
(NOS) amongst which iNOS (inducible NOS) is respon-
sible for apoptosis induction, oxidative stress and
DNA damage (Martin et al. 1999). In the current
study, there was an increase in the NO levels due to
P5 treatment which was simultaneous to the apopto-
sis of the cells indicating the activation of iNOS. This
increased NO level might also be contributing to the
observed cell death in cancer cells. According to an
earlier report, increased level of NOS was the major
cause for p53 accumulation, cell cycle arrest and
apoptosis (Hsieh et al. 1999).
The GC-MS analysis of P5 fraction, indicated the
presence of an indole-thiosemicarbazone deriva-
tive, with many reports indicating its anti-cancer
properties. The compound has been isolated from
plants belonging to Isatis genus, fruits from the
cannon ball tree Couroupita guinanensisAubl. and
Calanthe discolour Lindl., and was also found to be
secreted by the parotid gland of Bufo frogs. The
anti-cancer activity of these derivatives has been
well documented (Hall et al. 2009; Hussein et al.
2015; Ibrahim and Elsaman 2018). It can be
assumed that the presence of this compound
might be responsible for the observed anti-cancer
property of P5 in the current study.
10 P. VENKATACHALAM AND V. K. NADUMANE
To be an ideal anti-cancer agent, it is necessary that
the selected compound needs to be effective not only
against the cancer cell proliferation, but also should
be safe to the normal healthy cells. To verify the non-
toxicity of this promising compound, it was checked
on normal human peripheral lymphocytes and it was
found that P5 fraction was non-toxic to the lympho-
cytes at all the tested concentrations. When the total
cell count was taken through trypan blue assay (Table
S4), no significant changes were found. The LDH assay
results further confirmed the non-toxicity, as there
was negligible damage to the lymphocyte mem-
brane, thereby indicating its safety to the normal cells.
As many of the natural anti-cancer compounds in
clinical use today are associated with undesirable side
effects, finding an alternative compound with least
toxicities will be of great significance. The fraction P5
from Penicillium rubens JGIPR9 in the current study
offers such a promise towards drug development.
Acknowledgements
The authors are grateful to Prof. Leela Iyengar, Adjunct
Professor, JAIN (Deemed-to-be University) for her valuable
suggestions towards the research work conducted. The
authors are grateful to JAIN (Deemed-to-be University),
Bengaluru for providing the infrastructural facilities. The
authors would like to sincerely thank Dr R. Kavyashree,
Principal, Oxford College of Science, Bengaluru for permitting
to use the Nanodrop instrument for RNA quantification. The
authors would also like to earnestly thank Dr M.K. Prasanna
Kumar, Assistant Professor of Plant Pathology, GKVK, Bengaluru
for providing the RT-qPCR facility in his laboratory.
Author contributions
PV –Performed the research work and prepared the
manuscript
VKN –Planning, Supervising the research work and correcting
the manuscript
Both the authors have read and approved the manuscript
Data availability
The data that support the findings of this study are available
from the corresponding author (VKN), upon reasonable request.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research was financially supported by the Department of
Science and Technology –Innovation in Science Pursuit for
Inspired Research (DST- INSPIRE) Fellowship [grant no.
IF130879];Department of Science and Technology, Ministry of
science and technology, Government of India .
Informed consent
The written consent for blood sampling from human volun-
teers was collected and documented.
Research involving human participants
The blood sampling for lymphocyte isolation from human
volunteers was performed as per the ethical guidelines outlaid
by ICMR.
ORCID
Varalakshmi Kilingar Nadumane http://orcid.org/0000-
0001-9979-5007
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