1. 2 3 4 5 6 7 Cervical cancer Aim of The Present Study Lactobacilli Cervical cancer 8.
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Transcript of 1. 2 3 4 5 6 7 Cervical cancer Aim of The Present Study Lactobacilli Cervical cancer 8.
Cervical Cancer &Vaginal Lactobacilli
TUMS
Assistant professor
School of Advanced Medical Technologies,
Tehran University of Medical Sciences
Elahe Motevaseli
MD, Ph.D
1
Overview
•Introduction (Definition & Aims) •Materials & Methods•Results•Discussion
2
Introduction
3
Cervical Cancer, At a Glance
A slow-growing cancer that forms in tissues of cervix, the organ connecting uterus & vagina
Pre-malignant stages: Cervical Intraepithelial Neoplasia (CIN) 1, 2 and 3
Detect in regular Pap smear tests
The most frequently diagnosed female cancer in developing countries & the second most frequent cancer affecting women worldwide
Iran: 25.61 millions women >15 years at risk, every year 643 women diagnosed with cervical cancer & 286 die from the disease (WHO/ICO Information Centre, 2010)
4
Risk Factors
Human Papilloma Virus (HPV) infection: Bacterial vaginosis (BV) The other factors
90% of HPV infections resolve spontaneously The other factors: environmental & host factors,
cervical microbial flora & other infections seems to be necessary for the development of the disease.
5
Cervical Microbial Flora The healthy human vaginal & cervical ecosystem dominated by
Lactobacillus species Lactobacilli; sources of beneficial organisms termed Probiotics
What is Probiotics?
Live micro-organisms which confer a health benefit on the host when administered in adequate amounts
What do they do?• Modulate systemic inflammation, apoptosis & cell proliferation • Can control the overgrowth & infectious process of pathogens• Play an important role in maintenance of the normal vaginal flora by
inhibiting colonization of other pathogens.
6
hCGb, a Potential Cervical Cancer Biomarker
hCG, composed of two non covalently linked subunits – α (hCGa) & β (hCGb), physiologically produced by the placenta
Variety of tumors of different origins secrete hCGb
The presence of hCGb's mRNA & protein is a characteristic feature of cervical carcinomas, acts as an autocrine growth factor by inhibiting apoptosis (Jankowska et al., 2008)
Elevated serum level of hCGb correlates with an increased aggressiveness of cancer & its resistance to therapy
7http://www.genecards.org/ Jan 2012
Cervical cancer
Aim of The Present Study
Evaluation of the proliferative & apoptotic responses of normal & tumoral cervical cell lines to different components of two common vaginal lactobacilli (L. crispatus & L. gasseri)
LactobacilliBacterial
Vaginosis Cervical cancer
Other cancers
hCGb, Potential Cervical Cancer Biomarker
?
8
Materials & Methods
9
Experimental Procedures
Sampling
Gram staining (Nugent score):
Grade 1 (normal flora), Grade 2 (intermediate flora), Grade 3 (BV)
Bacterial diagnosis
Supernatant, cytoplasm, cell wall extracts
Cytotoxic assay:
Bacterial isolation Bacterial identification: Biochemical Molecular: 16s rRNA sequencing Multiplex PCR
Bacterial component separation
MTT assayTripan blue assayLDH assay
Expression analysis of βhCG by Real-Time PCR Biomarker Assay:
Apoptotic assay:
Caspase3 activity assayLDH assayReal-time PCR
10
Recruited at Gynecology Outpatient Clinic of Imam Khomeini Hospital (1386-1389)
Inform consent was obtained
Inclusion Criteria:
From 18 to 45 years (Reproductive age)
Exclusion Criteria:
Pregnancy
Menopause
Antibiotic or Antimycotic Compounds
Consumption
Sampling
Gram staining (Nugent score):
Bacterial diagnosis
Cytotoxic assay:
Bacterial component separation
Biomarker Assay:
Apoptotic assay:
11
Grade 1 (normal flora), lactobacillus morphotype only
Grade 2 (intermediate flora), reduced lactobacillus morphotype with mixed bacterial morphotypes
Grade 3 (BV), mixed bacterial morphotypes with few or absent lactobacillus morphotypes
Sampling
Gram staining (Nugent score) :(Ison et al., 2002)
Bacterial diagnosis
Cytotoxic assay :
Bacterial component separation
Biomarker Assay:
apoptotic assay:
Gra
de
1 G
rad
e 2
Gra
de
3
12
Sampling
Gram staining (Nugent score):
Bacterial diagnosis
Cytotoxic assay :
Bacterial component separation
Biomarker Assay:
apoptotic assay:
Biochemical identification API method(E.g. L-sorbose, D-fructose, D-galactose)
16S rRNA 23S rRNA
R
G I G III
G VIG II Molecular identification by multiplex PCR13
ISR
Bacterial colony isolation Bacterial identification: Biochemical: Gram (+)& catalase (-) Sugar fermentation (API) Molecular (next slide): 16s rRNA sequencing Multiplex PCR
Lactobacilli Molecular Identification, Primer Design For Multiplex PCR
16S rRNA 23S rRNA
Jensenii F
crispatus F crispatus R
gasseri R
acidophilus F
gasseri F
Jen , acid R
16S rRNA 23S rRNA
paracasei R rhamnosus Rpara , rham F
16S rRNA 23S rRNA
salivarius F
reuteri F
fermentum F
salivarius Rplantarum F
plantarum R
reuteri R
fermentum R
ISR
ISR
ISR
14
G III
G II
G VI
• L. crispatus strain SJ-3C-US (LbC) • L. gasseri ATCC 33323 (LbG)• L. rhamnosus GG• L. paracasei subsp. paracasei ATCC 25302 • L. casei var. Rhamnosus doderlein
(Gynophilus lyocentre)• L. acidophilus NCFM (probioti-NCF) • L. helveticus LA 401 candisis (Lactibiance
candisis 10M)
Sampling
Gram staining (Nugent score):
Bacterial diagnosis
Cytotoxic assay :
Bacterial component separation
Biomarker Assay:
Apoptotic assay:
Cell wall & Cytoplasmic Extract Separation
Colony Formation Unit (CFU) adjustment
Supernatant:• L. crispatus & L. gasseri supernatant
pH=4• MRS broth pH=6.5• MRS+HCl pH=4• MRS+Lactic acid pH=4• Lactobacilli supernatant+NaOH pH=6.5• Condition medium &live lactobacilli
cytoplasm & cell wall extracts:• Bacterial cell wall disruption
(homogenate) • Ultracentrifugation (separation)
Ultracentrifuge
16
Sampling
Gram staining (Nugent score):
Bacterial diagnosis
Cytotoxic assay :
Bacterial component separation
Biomarker Assay:Hela cell HNCF cell
Apoptotic assay:
17
• Hela cell(cervical cancer cell line) & HNCF-PI52 (normal cervical cell line)
MTT assay: (Colorimetric)
Mitochondria succinate dehydrogenase enzymes living cells
Reduce yellow water-soluble substrate (MTT) to insoluble, colored formazan product
• Optimal cell number determination
Tripan blue assay LDH assay
Sampling
Gram staining (Nugent score):
Bacterial diagnosis
Cytotoxic assay :
Bacterial component separation
Biomarker Assay:
Apoptotic assay:
18
Caspase3:• Synchronization• Treatment, Cell lysis • Protein concentration determination & adjustment• Caspase 3 activity assessment
Caspase 3 activity (%)=[(sample OD/ control OD)]×100
LDH:• LDH pellet: folating cells, LDH intra cellular: adherent cells, LDH extra
cellular: culture supernatant• Apoptosis (%) = [LDH pellet / LDH total] ×100
Necrosis (%) = [LDH extracellular / LDH total] ×100
Real-time PCR:• Caspase3, Fas, Bax, Bcl2, HPRT (housekeeping gene)
Sampling: 5 samples of cervical carcinoma, 5 samples of the uterine myometrium, 5 samples of normal cervix & 5 placentas as a control
RNA extraction of tissues & cell lines Expression analysis of βhCG by Real-Time RT-PCR
Sampling
Gram staining (Nugent score):
Bacterial diagnosis
Cytotoxic assay :
Bacterial component separation
Biomarker Assay:
Apoptotic assay:
Hallast P., Rull K.,Laan M. (2007) The evolution and genomic landscape of CGB1 and CGB2 genes. Mol Cell Endocrinol 260-262:2-11
20
hCGB Primer Design for Real-Time PCR
SYBR GREEN (Forward & Reverse)
Taqman (Forward & Reverse), probe (5’FAM-3’TAMRA)
Results
21
Grading of SpecimentsAccording to The Nugent Score
Number Nugent score
54 Grade I (Normal)
60 Grade II (Intermediate)
64 Grade III (BV infected)
178 Total
22
Multiplex PCR
Group I 450 bp
Group II 300 bpGroup III 400 bpGroup IV 350 bp
Group III multiplex PCR
Multiplex PCR Results for Lactobacilli Identification
Group III
L. paracasei 312bp
L. rhamnosus 113bp23
The Prevalence of Lactobacilli in Different Grades
Grade 1
Normal flora
No)%( .
Grade 2
intermediate
No)%( .
Grade 3
BV infected
No)%( .Multiplex PCR group I 0( 0) 0( 0%) 0( 0%)Lactobacillus delbrueckii 0( 0) 0( 0) 0( 0)Multiplex PCR group II 48( 88.9) 46( 76.7) 46( 71.9)Lactobacillus acidophilus 16( 29.6) 4( 6.7) 10( 15.6)
Lactobacillus crispatus 36( 66.7) 16( 26.7) 24( 37.5)Lactobacillus gasseri 16( 29.6) 6( 10) 14( 21.9)
Lactobacillus jensenii 16( 29.6) 14( 23.3) 6( 9.4)Multiplex PCR group III 24( 44.4) 38( 63.3) 46( 71.9)Lactobacillus paracasei 10( 18.5) 10( 16.7) 8( 12.5)Lactobacillus rhamnosus 16( 29.6) 24( 40) 30( 46.9)Multiplex PCR group IV 14( 25.9) 8( 13.3) 14( 21.9)L. salivarius 2( 3.7) 2( 3.3) 4( 6.3)L. reuteri 2( 3.7) 2( 3.3) 2( 3.1)L. plantarum 6( 11.1) 4( 6.7) 4( 6.3)L. fermentum 6( 11.1) 2( 3.3) 6( 9.4)L. iners 30( 55) 33(55) 50( 83)
P=0.007
P=0.047
P=0.01
MTT Results (Hela cell)
• Optimal cell determination for Hela cell
25
OD
Cell Number
Optimal cell determination for HNCF cell
MTT Results (HNCF cell)
26
Formazan crystals
OD
Cell Number
L.crispatus & L.gasseri Supernatant MTT Results
5%
Lactic acid + MRS pH= 4
HCl + MRS pH= 4
27Supernatants of: LGS: L.gasseri, LCS: L.crispatus, LGSN: LGS + NaOH, LCSN: LCS + NaOH, MRH: MRS + HCl, MRL: MRS + Lactic Acid, MRS :lactobacilli Media
5%
HNCF cell
Hela cell
via
bili
tyvi
ab
ility
Concentration (%)
Concentration (%)
Vaginal Lactobacilli & Commercial Probiotics Supernatant MTT Result
Supernatants of: LGS: L.gasseri LCS: L.crispatus LRS: L.rhamnosus LPS: L.paracasei LHS: L.helveticus LNS: L.acidophilus NCFM LAS: L.acidophilus candisis and MRS :lactobacilli Media as control
Commercial Probiotics
Commercial Probiotics
28
pH=4 pH=5 pH=6.5
Hela cell
HNCF cell
via
bili
ty
Concentration (%)
via
bili
ty
Concentration (%)
Homogenate, Cell Wall & Cytoplasmic Extract MTT Results
29
Hela cell
via
bili
ty
Concentration (%)
LDH & Trypan Blue Confirmed The MTT Assay Result
Live lactobacilli effect:Inhibitory effect on Hela cells but no effect on HNCF cells
Conditioned media effect: No cytotoxic effect on both cell lines
2
5
10
20
50
MRSLCS
30
LDH Assay
Apoptotic Assay, Caspase3 Activity Assay
LGS & LCS effect:Caspase3 activity reduction
31
Apoptotic inhibition of lactobacilli supernatants is independent of pH & lactic acid
MRS & MRL effect:No change in caspase3 activity
LDH Assay; Apoptotis & Necrosis Ratio
The ratio of LDH released from adherent cells, floating dead cells & the culture supernatant were compared:
.
32
Lactobacilli supernatants lowered the number of apoptotic cells of Hela cells, independent of pH & lactic acid
Hela cell HNCF cell
Real-time PCR Results (Quality Controls)
ºC65 70 75 80 85 90 95
dF/d
T
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
ºC65 70 75 80 85 90 95
dF/dT
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
deg.65 70 75 80 85 90 95
dF/dT
11
10
9
8
7
6
5
4
3
2
1
0
deg.65 70 75 80 85 90 95 100
dF/dT
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
HPRTMP=78°C
Bcl2 MP=87°C
Fas MP=81°C
Bax MP=87°C
Caspase3 MP=81°C
Neg
Bcl2: 127bp HPRT:131bp
La
dd
er:
10
0b
p33
Gel Electerophoresis; only for the first time
Melting Point Analysis; after each Real-Time PCR run
Real-time PCR Results Expression Analysis of Bax & Bcl2,
(Involved in Intrinsic Apoptotic Pathway)
34
LGS 10% LCS 10% MRS 10%0.70
0.81 0.79
1.02
lactobacilli Supernatant Effect on Bax Expression in Hela Cell
Ab
so
lute
Gen
e R
eg
u-la
tio
nLo
gari
thm
ic S
cale
LGS 10% LCS 10% MRS 10%0.70
1.12 1.15
0.80
lactobacilli Supernatant Effect on Bcl2 Expression in Hela Cell
Ab
so
lute
Gen
e R
eg
u-la
tio
n
Lo
gari
thm
ic S
cale
P= 0.35P= 0.66
P= 0.39
P= 0.73
P= 0.67P= 0.39
35
LGS 10% LCS 10% MRS 10%0.70
0.92
1.02
0.84
Lactobacilli Supernatant Effect on Fas Expression in Hela Cell
Ab
solu
te G
en
e R
eg
ula
tio
nLo
gari
thm
ic S
cale
Real-time PCR Results Expression Analysis of Fas,
(Involved in Extrinsic Apoptotic Pathway)
P= 0.35 P= 0.34P= 0.84
36
LGS 10% LCS 10% MRS 10%0.30
0.36 0.50
1.36
Lactobacilli Supernatant Effect on Caspase3 Expression in Hela Cell
Ab
solu
te G
en
e R
eg
ula
tio
nLo
gari
thm
ic S
cale
Real-time PCR Results Expression Analysis of Caspase3,
(Activated by Both Extrinsic & Intrinsic Pathways)
P= 0.001 P= 0.001
P= 0.31
Biomarker Assay; Expression Analysis of hCGb by ReaL-Time PCR
37
ºC65 70 75 80 85 90 95
dF/dT
1.0
0.8
0.6
0.4
0.2
0.0
hCGbMP= 89°C
Tissue Cgb expression
placenta +
Cervical cancer +
Hela +
Uterine myometrium -
Normal cervix -
38
Real-time PCR Results Expression Analysis of cgb,
(Potential Cervical Cancer Biomarker)
LGS 10% LCS 10% MRS 10%0.60
1.53
1.34
0.63
Lactobacilli Supernatant Effect on cgb Expression in Hela Cell
Ab
solu
te G
en
e R
eg
u-la
tion
Log
ari
thm
ic S
cale
P= 0.29P= 0.04
P= 0.001
LGS LCS MRS0.3
LGS & LCS Effect on cgb & Caspase3 Expression
caspase3cgb
Ab
solu
t G
en
e R
eg
ula
tion
Log
ari
thm
ic S
cale
P= 0.001
P= 0.04P= 0.29
P= 0.001
P= 0.001
P= 0.001
P= 0.31
Caspase3 & cgb Expression,(Cytoplasmic Extract & Cell Wall)
39
No significant change was observed
Discussion
40
Snapshot of ResultsThe Prevalence of Lactobacilli in Different Grades: (biochemical i & Multiplex PCR) Some lactobacilli were more prevalent in vaginal flora of healthy womenL. crispatus & L. jensenii higher in healthy women & L. iners higher in BV infected
Bacterial Component Separation; Searching For Main Effectors
41
Cytotoxic Effects Apoptotic Effects
Lactobacilli Supernatants: Most potent cytotoxic effects Lactic acid production >>pHTumor cells >>>Normal cellsNormal cells: Supernatant effect = MRL effect but Tumoral cells: supernatant effect ≠ MRL effect L. crispatus & L. gasseri effect >commercial vaginal probiotics effect
Homogenate, Cell Wall & Cytoplasmic Extract:Homogenate effect = cytoplasmic extract effect < supernatant effectCell wall; no effect
Live Lactobacilli: Inhibitory effect on tumor cells but no effect on normal cells Conditioned Media Effect: No cytotoxic effect
Lactobacilli Supernatants: Anti-apoptotic effect on tumoral cells but not on normal cells Lactic acid independentIntrinsic Apoptotic Pathway: Bax & Bcl2 (not signifacant)Extrinsic Apoptotic Pathway: Fas expression not changed (not signifacant)both Extrinsic & Intrinsic Pathways: Caspase3 Expression (signifacant)Biomarker assay: cgb as potential cervical cancer biomarker, overexpression by lactobailli supernatant, consistent with apoptotic inhibition
Prevalence of Vaginal Lactobacilli of Healthy Women; Similarities Between Our Study & Recent Findings
• In the past:
L. acidophilus,
L. fermentum, L. brevis,
L. Jensenii & L. casei
(Lachlak et al.,1996)
• Recent studies:
L. crispatus, L. gasseri,
L. iners, & L. jensenii
(Ravel et al., 2011)
Our results: L. crispatus, L. gasseri, L. iners, L. jensenii, L.acidophilus & L. rhamnosus
42
Comparison of Healthy Vaginal Flora With BV Infected
in Different Studies & Our Study
• Japan: (Tamrakar et al., 2007) L. iners higher in BV infected
• Belgium: (Verstraelen et al., 2009) L. crispatus, higher in healthy. L. gasseri & L. iners higher in BV
infected
• South Africa: (Damelin et al., 2011) L.crispatus distributed equally between healthy & BV infected.
L. jensenii higher in healthy
Our results: L. crispatus & L. jensenii higher in healthy women & L. iners higher in BV infected
43
Most Potent Cytotoxic Component
• Sekine et al.1995: peptidoglycans from
B. infantis have anti-tumor activity
• Orlando et al. 2009:
Cytoplasm fraction from Lactobacillus rhamnosus GG induced an anti-proliferative effect whereas cell wall fractions had no significant effect
Our study: lactobacilli supernatants have most potent cytotoxic effects rather than other components
44
Lactic Acid Effect - The pH EffectsWhich One?!
• Some suggested that Probiotics activities resulted from their lactic acid production & pH of their cultures
• MRL (MRS acidified with lactic acid) showed more potent inhibiting effect on both cell lines rather than MRH (MRS acidified with hydrochloric acid), although they had similar pH
• lactic acid production; more important part of lactobacilli inhibitory effect than pH alone
45
Cancer Cell Selective Effects
• Choi et al., 2006:
L. acidophilus inhibits cancer cell proliferation but less effect on normal cells
Our study:
• Lactobacilli supernatants had cytotoxic effects on tumor cells but less effect on normal cells.
• Normal cells (HNCF) responded to the supernatants similar to MRL
(contain Lactic acid) but tumor cells (Hela) responses to supernatants & MRL were clearly different.
• There are non lactate molecules in lactobacilli supernatants which have anti-cancer activities but they are safe for normal tissues. 46
Anti-apoptotic Effect of Lactobacilli Supernatants
• Choi et al. 2006: soluble polysaccharides from L. acidophilus resulted in the death of cancer cells by the induction of apoptosis
• Khailova et al. 2010: Bifidobacterium bifidum reduces apoptosis in the intestinal epithelium in necrotizing enterocolitis
• Sharma et al. 2011: probiotic cell lysate administration; a promising approach for reducing mitochondria mediated oxidative stress & subsequent apoptosis
47
Our study:lactobacilli supernatants have anti-apoptotic effect on Hela cells
This anti-apoptotic effect was significantly lactic acid independent.
Lactobacilli supernatant
Apoptosis
hCGb
MKP1
Autophagy TGF β
receptor Cell death
Puzzle Arrangement (Our Study & Other Studies)
48
(Kim et al., 2010)
(Brion et al., 2011) (Brion et al., 2011)
(Brion et al., 2011)
(Brion et al., 2011)
(Vermeulen et al.,2010)
(Del Canto et al., 2007)
(Iles, 2007)(Tsukamoto et al., 2008)
(Iles, 2007)
(Khailova et al.2010)Our finding: Anti-apoptotic effect of lactobacilli & over-expression of hCGb
Apoptosis Inhibits Autophagy
49(Kang et al., 2011)
Suggestions•L. crispatus strain SJ-3C-US & L. gasseri ATCC 33323 more cytoxicity effect on tumoral cervical cells compared to commercial vaginal probiotics; recommended as anti-cancer probiotics
•To separate different components of lactobacilli supernatants & identify non lactate molecules with selective anticancer activities, High Performance Liquid Chromatography (HPLC) is recommended
•As autophagy would be the cell death mechanism of lactobacilli supernatants, expression analysis of genes affecting autophagic pathway as Beclin1 is recommended
•The assessment of the relation between lactobacilli & HPV is recommended
•The potency of hcgb to be considered as a biomarker in cervical cancer; treatment, outcome evaluation & cancer staging
• Vaginal flora lactobacilli determination could as a reliable indicator for susceptibility assessment
50
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