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British Journal of Healthcare and Medical Research - Vol. 11, No. 5
Publication Date: October 25, 2024
DOI:10.14738/bjhmr.115.17644.
Gonen, C. (2024). PMA Induction to Prostate Cancer Cell Lines: DU145 and LNCaP. British Journal of Healthcare and Medical
Research, Vol - 11(5). 86-94.
Services for Science and Education – United Kingdom
PMA Induction to Prostate Cancer Cell Lines: DU145 and LNCaP
Ceren Gonen
Ege Univ. Pharmacy Fac. Pharmacology Dept.Bornova, Izmir, Turkiye
ABSTRACT
Prostate cancer is the most commonly diagnosed cancer and the second leading
cause of cancer mortality in men in the Western World. The effects of androgens are
mediated by the Androgen Receptor (AR). Therefore, studies focus on the
identification of AR-regulated genes that are also highly expressed in the prostate.
STAMP family genes STAMP1/STEAP2 and STAMP2/STEAP4 have only expressed in
androgen receptor-positivecells, the role of AR in STAMP family gene expression is
an important question Phorbol 12-myristate 13-acetate, NF-κB Activator, PMA, TPA
PMA is used at 100nM final concentration after transfection of Hismax vector and
Hismax- STAMP1 to DU145-brain metastases of prostate cancer cell line. DU145 cell
line kinetics were shown. PMA induction to LNCaP cells.was done. In summary, the
data presented showthat STAMP1 is involved in a signaling pathway that is linked
to cell cycle progression as well as inhibition of apoptosis. Consistent with its
increased expression in prostate cancer, these data suggest that STAMP1 may play
important roles in prostate cancer development andprogression.
Keywords: DU145, LNCaP, PMA, RT-PCR, STAMP1, AR: Androgen receptor, ARRE:
Androgen receptor response element, LNCaP Lymph Node Metastases Prostate Cancer
Cell Line, PMA: Forbol Miristal Asetat, STAMP: six transmembrane protein of prostate,
RTqPCR: Quantitative polymerase chain reaction TUBA the Turkish Academy of
Sciences, TUBITAK The Scientific and Technological Research Council of Turkey
INTRODUCTION
STEAP (Six Transmembrane Epithelial Antigens of Prostate) is the first characterized prostate
enriched six transmembrane genes, expressed in metastatic prostate cancer samples, it is
tempting to speculate that STAMP/STEAP family genes may be involved in similar functions
with a role for both the normal biology and pathophysiology of the prostate.
Phorbol 12-myristate 13-acetate (PMA), also known as 12-O-tetradecanoylphorbol 13- acetate
(TPA), is a specific activator of Protein Kinase C (PKC) and hence activates nuclear factor-kappa
B (NF-κB). NF-κB is a transcription factor that regulates numerous physiological functions and
is involved in the pathogenesis of various diseases. It has been identified as a potential
therapeutic target in inflammatory processes, cancer, and autoimmunediseases.
PMA is the most common and potent phorbol ester. It is active at nanomolar concentrationsand
activates NF-κB in a dose-dependent manner. PMA causes a wide range of effects in cells and
tissues and is a very potent mouse skin tumor promoter.
InvivoGen’s PMA is designed to study the NF-κB pathway in cellular assays.
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Gonen, C. (2024). PMA Induction to Prostate Cancer Cell Lines: DU145 and LNCaP. British Journal of Healthcare and Medical Research, Vol - 11(5).
86-94.
URL: http://dx.doi.org/10.14738/bjhmr.115.17644.
Because knockdown of STAMP1 increased cell death, it was of interest to investigate if there
were changes in STAMP1 levels in prostate cancer cells that are undergoing apoptosis.
Previous studies have established that apoptosis in LNCaP cells can be induced by various
agents. Prostate cancer is cancer of the prostate. The prostate is a gland in the male
reproductive system that surrounds the urethra just below the bladder. Most prostate cancers
are slow growing. Cancerous cells may spread to other areas ofthe body, particularly bones and
lymph nodes. It may initially cause no symptoms. In later stages, symptoms include painor
difficulty urinating, blood in the urine, or pain in the pelvis or back. Benign prostatic hyperplasia
may produce similar symptoms. Other late symptoms include fatigue, due to lowlevels of red
blood cells.
Factors that increase the risk of prostate cancer include older age, family history, and race.
About 99% of cases occur after age 50. A first-degree relative with the disease increases therisk
two- to three-fold. Other factors include a diet high in processed meat and red meat, while the
risk from a high intake of milk products is inconclusive.
NF-kappa B is a transcription factor found in all cell types. It is inactive in the cytoplasm. When
activated, it is transported to the nucleus. There are 5 types: NF-kB1, NF-kB2, RelA,RelB, and c- Rel. It is thought that NF-kappa B has an effect in some autoimmune diseases.
RESULTS
PMA used at 100nM final concentration after transfection of Hismax vector (1A) andHismax- STAMP1 (1B) to DU145-brain metastases of prostate cancer cell line.
1A DU145 cells transfected wirh HisMax-vector pMAinduced RT-PCR primers amplified.
Figure 1 A: DU145 cells transfected wirh HisMax-vector PMAinduced RT-PCR primersamplified.
1B DU145 cells transfected wirh HisMax-STAMP1 PMAinduced RT-PCR primersamplified.
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Services for Science and Education – United Kingdom
Figuree 1B: DU145 cells transfecred with HisMax-STAMP1 PMA induced RT-PCR primers
amplified
PMA induction to HisMax vector transfected DU145 cells caused mouse double minute gene2-
MDM2 –decrease p<0.001 (1A)
But transfection of STAMP1 changed PMA responses and Akt1, BAD, CASPASE9 and, P73
showed 5.6, 3.9, 3.3, 2. 9 fold increase. respectevly p<0.001 (1B).
1C: DU145 cell line kinetics: HisMax-Vector- HisMax-STAMP1, HisMax-STAMP2 andHisMax- STAMP3 transfected
Figure1C: DU145 cells transfected with STAMP constructs (STAMP1, 2 and 3 versusHisMax- vector) growth kinetic result
DU145 cells transfected with STAMP constructs (STAMP1, 2 and 3 versus HisMax- vector)
STAMP/STEAP genes transfection caused gradual growth in brain metastasis of prostate cancer
cell line-DU145 cells. Following the day of the transfection done with FuGene HD transfection
reagent, cells were taken to 12-well plates as 104 cells per well and dead cells were counted by
trypan-blue exclusion assay beginning at day 2 until 12. Six time points weretaken that assayed
as n=7 per point and growth kinetics were drawn (Figure 1C). It was shown that STAMP3
transfection caused relatively lower growth increase compare to control group in the
transfected cell group.
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Gonen, C. (2024). PMA Induction to Prostate Cancer Cell Lines: DU145 and LNCaP. British Journal of Healthcare and Medical Research, Vol - 11(5).
86-94.
URL: http://dx.doi.org/10.14738/bjhmr.115.17644.
PMA induction to LNCaP cells was done.
Caspase7 and 9 showed a decrease P<0.001 and MDM2 gave an incraese p<0.001(FIG2),
Figure 2: LNCaP cells induced wirh PMA, RT-PCR primers amplified
DISCUSSION
p53 or tumor protein 53 (TP53), Genome Guardian, anti-tumor p53 is a transcription factor
that regulates the cell cycle. It is a very important protein for suppressing cancer in many
organisms. It is critical as it inhibits cancer formation in multicellular vertebrates and exhibits
tumor suppressive function.
p73 is a protein associated with the p53 tumor protein. Because of its structural similarity to
p53, it has also been considered a tumor suppressor. It plays a role in cell cycle regulation and
apoptosis induction. Like p53, p73 is characterized by the presence of different isoforms of the
protein.
The murine double minute (MDM2) oncogene, which codes for the MDM2 protein, was
originally cloned, along with two other genes (MDM1 and MDM3) from the transformed mouse
cell line 3T3-DM. MDM2 overexpression, in cooperation with oncogenic Ras, promotes
transformation of primary rodent fibroblasts, and MDM2 expression led to tumor formation in
nude mice. The human homologue of this protein was later identified and is sometimes called
HDM2. Further supporting the role of MDM2 as an oncogene, several human tumor types have
been shown to have increased levels of MDM2, including soft tissue sarcomas and
osteosarcomas as well as breast tumors. The MDM2 oncoprotein ubiquitinates and antagonizes
p53, but may also carry out p53-independent functions. MDM2 supports the Polycomb- mediated repression of lineage-specific genes, independent of p53. MDM2 depletion in the
absence of p53 promoted the differentiation of human mesenchymal stem cells and diminished
clonogenic survival of cancer cells. Most of the MDM2-controlled genesalso responded to the
inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2.
MDM2 physically associated with EZH2 on chromatin, enhancing the trimethylation of histone
3 at lysine 27 (H3K27me3) and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its
target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2
further induced these genes and synthetically arrested cell proliferation.
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AKT1
AKT1 (gene at 14q32.33) is one of three closely related serine/threonine-protein kinases
(AKT1, AKT2, and AKT3) that regulate many processes including metabolism, proliferation, cell
survival, growth, and angiogenesis by phosphorylating a range of downstream substratesin
response to growth factor stimulation (i.e., EGF, IGF). AKT is responsible of the regulation of
glucose uptake by mediating insulin-induced translocation of the SLC2A4/GLUT4 glucose
transporter to the cell surface. It also regulates the storage of glucose in the form of glycogen.
AKT promotes cell survival via the phosphorylation of the apoptosis signal-related kinase
MAP3K5. Phosphorylation of “Ser-83” decreases MAP3K5 kinase activity stimulated by
oxidative stress and thereby prevents apoptosis. AKT has an important role in the regulation of
NF-kappa-B-dependent gene transcription and positively regulates the activity of the cyclic
AMP (cAMP)-response element binding protein (CREB1). The phosphorylation of CREB1
induces the binding of accessory proteins that are necessary for the transcription ofpro-survival
genes.
BAD: Bcl2 Antagonist of Cell Death
Yang et al. (1995) identified a new protein that binds to Bcl2. Antagonist of Bcl2-mediated cell
death: It is named Bcl2 antagonist of cell death: BAD. ' This 204-amino acid protein has
sequence similarity to the BH1 and BH2 domains of BCL2 family members. Bad shows strong
heterodimerization with BclX from this family, weaker binding with Bcl2 but no binding with
other members. In functional studies, it has been shown that Bax is removed as aresult of
dimerization of Bad with BclX and its apoptosis-inducing effect is eliminated.
BNIP
BNip (formerly known as Nip) proteins, including homologues isolated from human, mouse and
Caenorhabditis. elegans, are a relatively new subgroup of the Bcl-2 family. These proteins are
classified into this family based on limited sequence homology with the Bcl- 2homology domain
3 and carboxyl terminal transmembrane domain. BNip proteins were firstdiscovered based on
their interaction with the adenovirus E1B 19 kDa/Bcl-2 family protein and since then, their
roles in cell death pathways have been actively studied. However, the precise mechanisms by
which the BNip proteins induce apoptosis and/or necrosis remain to be determined.
Caspase7
The described roles of caspase-7 in apoptosis and inflammation suggest that interfering with
caspase-7 activation may prove beneficial in conditions where excessive cell death and/or
inflammation contribute to disease. Therapeutic inhibition can be achieved with synthetic
inhibitors or through targeted delivery of natural caspase inhibitors such as XIAP and the
baculoviral caspase inhibitor p35. Caspase-7 inhibition seems especially warranted in
neurodegenerative disorders such as Alzheimer’s disease and Huntington’s disease, where
increased caspase-7 expression correlates with excessive neuronal cell death. Another
potentially promising application is the prevention of lymphocyte cell death in sepsis. Extensive
leukocyte apoptosis is commonly observed in sepsis patients and was suggested tocontribute
to immune suppression and lethality). Synthetic caspase inhibitors and overexpression of the
anti-apoptotic protein Bcl-2 have already shown promising results in experimental sepsis
models). In addition to the potential roles of caspase-7 in sepsis and neurodegenerative
disorders, single nucleotide polymorphisms (SNPs) in the caspase-7 genehave been linked with
rheumatoid arthritis (SNP rs2227309; K249R mutation) Insulin- Dependent Diabetes Mellitus
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Gonen, C. (2024). PMA Induction to Prostate Cancer Cell Lines: DU145 and LNCaP. British Journal of Healthcare and Medical Research, Vol - 11(5).
86-94.
URL: http://dx.doi.org/10.14738/bjhmr.115.17644.
(SNP rs144692; D251E mutation) Additional studies are required to extend these promising
findings and to unravel the mechanisms by which the resulting missense mutations in caspase- 7 trigger disease Caspase 9.
Caspase 9 (also known as ICE-LAP6, or mch6) is considered to be a caspase in the upstream of
apoptotic signal transduction. When mitochondria release cytochrome c, Caspase 9 can form
complex with cytochrome c and Apaf1, and be activated at the same time. Activated Caspase 9
can activate Caspase 3 which is the key enzyme of cell apoptosis, thereby promoting subsequent
apoptotic signals. Caspase 9 exists in the form of prozyme in the normal state and has no
activity. However, Caspase 9 is activated in the apoptotic stage and participates in the apoptotic
process. This kit is used to conjugate Caspase 9 sequence-specificpeptides acetyl-Leu-Glu-His- Asp p-nitroanilide (Ac-LEHD-pNA) to yellow group p- nitroaniline (pNA)
Many of the roles played by the tumor suppressor in restraining cancer initiation and
progression are well established. These include the ability of to induce cell-cycle arrest, DNA
repair, senescence and apoptosis One of the hallmarks of human cancers is the intrinsic or
acquired resistance to apoptosis. Evasion of apoptosis can be part of a cellular stress response
to ensure the cell’s survival upon exposure to stress stimuli. There are two major apoptosis
signaling pathways, that is, the dead receptor (extrinsic) pathway and the mitochondrial
(intrinsic) pathway]. Under most circumstances, activation of either pathway eventually leads
to proteolytic cleavage and thus activation of caspases, a family of cysteine proteases that act
as common dead effector molecules. Accordingly, caspases are responsible for many of the
biochemical and morphological hallmarks of apoptotic cell dead by cleaving a range of
substrates in the cytoplasm or nucleus Cytochrome c has been implicated in the redox
regulation of apoptosis. Once cytochrome c is released from mitochondria into the cytosol, it
triggers the formation of the cytochrome c/Apaf-1/Caspase-9-containing apoptosome, which
in turn lead to activation of caspase-9 and downstream effector caspases There is recent
evidence that also the redox state of cytochrome c is involved in the regulation of apoptosis. To
this end, the oxidized form of cytochrome c (Fe(3+)) has been reported to induce caspase
activation via the apoptosome, while the reduced form of cytochrome c (Fe(2+)) is unable to do
so Response elements of prostate specific antigen (PSA) and NFkB are located at AR promoter
region and suggest that NFkB may affect AR expression.
Statistical Analysis
All the illustrated results represent one of at least three independent experiments withsimilar
outcomes.
Acknowledgement
This work was granted by The Scientific and Technological Research Council of Turkey
(TUBITAK) to CGK [Grant no: 106S295]; The Turkish Academy of Sciences (TUBA) to CGK
[GEBIP-2007].
Experimental Procedures Cell Culture
Cell lines were cultured in DMEM (GIBCO, USA) or RPMI 1640 (GIBCO) with 5% and 10% fetal
bovine serum for DU145 and LNCaP cells, respectively. 1% L-glutamine and 1U/ml of each
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penicillin/ streptomycin was used. Cells were incubated at 37°C and 5% CO2in a humidified
atmosphere.
TABLE 1: Primers for RT-PCR amplification
GeneBank/
Symbol
Description Gen Name Primers
NM_004322
BAD
BCL2-antagonist of cell
death
BBC2/BCL2L8 Forward
Reverse
AGGATCCGTGCTGTCTCCTTTG
CAAAACTTCCGATGGGACCAAG
NM_001188 BCL2-antagonist
/killer 1
BAK/BCL2L7 Forward GGGTGTAGATGGGGGAACTGTG
BAK1 Reverse AAGACCCTAGGCTGTGCCCAAT
NM_138578 BCL2-like 1 BCL-X/BCL-XL Forward GTGTGAGGAGCTGCTGGCTTG
BCL2L1 Reverse AGCATCAGGCCGTCCAATCTC
NM_001205
BNIP1
BCL2/adenovirusE1B 19kDa
interacting
protein 1
NIP1/TRG-8 Forward
Reverse
CAGGTTGGATGGAACACAGTGC
ATCCCAATGCCAGACCTTCCTC
NM_032982
CASP2
Caspase 2,
apoptosisrelated cysteine
protease
CASP-2/ICH-1L Forward
Reverse
TCTCCCATGGTCCCTAGCAAAA
AAGGCTCACAAACCACCCAAAC
NM_001227 Caspase 7,
apoptosisrelated cysteine
protease
CMH-1/ICE-LAP3 Forward AAGTGAGGAAGAGTTTATGGCAAA
CASP7 Reverse CCATCTTGAAAACAAAGTGCCAAA
NM_001229
CASP9
Caspase 9, apoptosisrelated
cysteine protease
APAF-3/APAF3 Forward
Reverse
TCCTGAGTGGTGCCAAACAAAA
AGTGGTTGTCAGGCGAGGAAAG
NM_005157
ABL1
V-abl Abelson murine
leukemiaviral oncogene
homolog 1
ABL/C-ABL Forward
Reverse
GGCCTTGAAGACAGAGCAAAGC
GGAAGGGACCAGTACCTCATGG
NM_005163
AKT1
V-akt murine thymoma viral
oncogene
homolog 1
PKB/PRKBA Forward
Reverse
TCCCCCTCAGATGATCTCTCCA
CGGAAAGGTTAAGCGTCGAAAA
NM_005427 Tumor proteinp73 P73 Forward AGCAGCCCATCAAGGAGGAGTT
TP73 Reverse TCCTGAGGCAGTTTTGGACACA
NM_000546
TP53
Tumor protein p53
(LiFraumeni
syndrome)
CYS51STOP/P53 Forward
Reverse
AGATGGGGTCTCACAGTGTTGC
ATGTTGACCCTTCCAGCTCCAC
NM_078467
P21
Homo sapiens
cyclindependent kinase
inhibitor
1A
CDKN1A Forward
Reverse
GGCAGACCAGCATGACAGATT
GCGGCCAGGGTATGTACATGA
NM_002392
MDM2
Homo sapiens Mdm2,
transformed 3T3cell double
minute 2
HDMX/MGC71221 Forward
Reverse
GGGTTCGCACCATTCTCCTG
GGCAGATGACTGTAGGCCAAGC
NM_016335 Homo sapiens
proline
PIG6/HSPOX2 Forward TTTTTCACCCCACACTTGCAGA
TGTCCCAGGCAGGTATCAGGTT
PRODH dehydrogenase
(oxidase) 1
Reverse
NM_001101 Homo sapiens actin, beta PS1TP5BP1, beta- actin
Forward CAATGTGGCCGAGGACTTTGAT
ACTB Reverse AGTGGGGTGGCTTTTAGGATGG
NM_002046
GAPDH
Homo sapiens
glyceraldehyde 3-phosphate
dehydrogenase
G3PD, GAPD Forward
Reverse
CATTGCCCTCAACGACCACTTT
GGTGGTCCAGGGGTCTTACTCC
References
BRUCKHEIMER, E. M. and Kyprianou, N. Apoptosis in prostate carcinogenesis. A growthregulator and a
therapeutic target. Cell Tissue Res, 301(1), 153-62 (2000).