TUMS
Cervical Cancer &Vaginal Lactobacilli
Elahe Motevaseli
MD, Ph.D
Assistant professor
School of Advanced Medical Technologies,
Tehran University of Medical Sciences
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
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
Cervical cancer
http://www.genecards.org/
Jan 2012
7
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)
?
Lactobacilli
Cervical
cancer
hCGb, Potential Cervical Cancer Biomarker
8
Materials & Methods
9
Experimental Procedures
Sampling
Gram staining
(Nugent score):
Bacterial diagnosis
Bacterial component
separation
Cytotoxic assay:
Apoptotic assay:
Biomarker Assay:
Grade 1 (normal flora), Grade 2 (intermediate
flora), Grade 3 (BV)
Bacterial isolation
Bacterial identification:
Biochemical
Molecular: 16s rRNA sequencing
Multiplex PCR
Supernatant, cytoplasm, cell wall extracts
MTT assay
Tripan blue assay
LDH assay
Caspase3 activity assay
LDH assay
Real-time PCR
Expression analysis of βhCG by Real-Time PCR
10
Sampling
Gram staining
(Nugent score):
Bacterial diagnosis
Bacterial component
separation
Cytotoxic assay:
Apoptotic assay:
 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
Biomarker Assay:
11
Sampling
Grade 1
Gram staining
(Nugent score) :(Ison et al., 2002)
Grade
1 diagnosis
(normal flora), lactobacillus morphotype only
Bacterial
Bacterial component
Grade 2
Grade
2 (intermediate flora), reduced lactobacillus
separation
morphotype with mixed bacterial morphotypes
Cytotoxic assay :
Grade 3
Grade 3 (BV), mixed bacterial morphotypes with few
apoptotic
assay:lactobacillus morphotypes
or absent
Biomarker Assay:
12
Sampling
Gram staining
(Nugent score):
 Bacterial colony isolation
 Bacterial identification:
Bacterial diagnosis
Biochemical:
Gram (+)& catalase (-)
Sugar fermentation (API)
Bacterial component
Molecular (next slide):
separation
16s rRNA sequencing
Cytotoxic assay
:
Multiplex
PCR
ISR
apoptotic assay:
16S rRNA
Biochemical identification API method
(E.g. L-sorbose, D-fructose, D-galactose)
23S rRNA
GI
G Assay:
III
Biomarker
G VI
G II
R
Molecular identification by multiplex PCR
13
Lactobacilli Molecular Identification, Primer Design
For Multiplex PCR
ISR
16S rRNA
23S rRNA
Jensenii F
G II
crispatus F
acidophilus F
gasseri F
Jen , acid R
crispatus R
gasseri R
16S rRNA
ISR
para , rham F
23S rRNA
paracasei R
rhamnosus R
G III
ISR
16S rRNA
23S rRNA
salivarius F
G VI
reuteri F
plantarum F
fermentum F
reuteri R
salivarius R
plantarum R
fermentum R
14
Sampling
Gram staining
(Nugent
Colony
Formation
score):
Unit (CFU)
adjustment
diagnosis
Bacterial
Supernatant:
•
L. crispatus & L. gasseri
supernatant pH=4
•Bacterial
MRS component
broth pH=6.5
•separation
MRS+HCl pH=4
• MRS+Lactic acid pH=4
assay :supernatant+NaOH
•Cytotoxic
Lactobacilli
pH=6.5
• Condition medium &live lactobacilli
•
•
•
•
•
•
•
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)
assay: & cell wall extracts:
Apoptotic
cytoplasm
•
Bacterial cell wall disruption
(homogenate)
Assay:
•Biomarker
Ultracentrifugation
(separation)
Cell wall & Cytoplasmic
Extract Separation
Ultracentrifuge
16
Sampling
•
Gram staining
(Nugent
Hela score):
cell(cervical
cancer cell line) & HNCF-PI52 (normal cervical cell line)
diagnosis
Bacterial
MTT assay:
(Colorimetric)
Mitochondria succinate dehydrogenase enzymes living cells
Reduce yellow water-soluble substrate (MTT) to insoluble, colored formazan
Bacterial component
product
separation
• Optimal cell number determination
Cytotoxic assay :
 Tripan blue assay
 LDH assay
Apoptotic assay:
Biomarker Assay:
Hela cell
HNCF cell
17
Sampling
 Gram
Caspase3:
staining
•
•
•
•
(Nugent
score):
Synchronization
Treatment, Cell lysis
Bacterial
Proteindiagnosis
concentration determination & adjustment
Caspase 3 activity assessment
Caspase 3 activity (%)=[(sample OD/ control OD)]×100
Bacterial component
separation
 LDH:
Cytotoxic assay :
• LDH pellet: folating cells, LDH intra cellular: adherent cells, LDH extra
cellular: culture supernatant
• Apoptosis (%) = [LDH pellet / LDH total] ×100
Apoptotic assay:
Necrosis (%) = [LDH extracellular / LDH total] ×100
Assay:
 Biomarker
Real-time
PCR:
•
Caspase3, Fas, Bax, Bcl2, HPRT (housekeeping gene)
18
Sampling
Gram staining
(Nugent score):
 Sampling: 5 samples of cervical carcinoma, 5 samples of the uterine
myometrium,
Bacterial
diagnosis 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
Bacterial component
separation
Cytotoxic assay :
Apoptotic assay:
Biomarker 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
hCGB Primer Design for Real-Time PCR
 SYBR GREEN (Forward &
Reverse)
 Taqman (Forward & Reverse), probe
(5’FAM-3’TAMRA)
20
Results
21
Grading of Speciments
According 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 Results for Lactobacilli Identification
Multiplex PCR
Group I
450bp
Group II
300bp
Group III
400bp
Group IV
350bp
Group III multiplex PCR
Group III
L. paracasei
312bp
L. rhamnosus
113bp
23
The Prevalence of Lactobacilli in Different Grades
Grade 1
Grade 2
Grade 3
Normal flora
intermediate
BV infected
Multiplex PCR group I
No. (%)
0 (0)
No. (%)
0 (0%)
No. (%)
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
OD
Cell Number
25
MTT Results (HNCF cell)
Optimal cell determination for HNCF cell
Formazan crystals
OD
Cell Number
26
L.crispatus & L.gasseri Supernatant MTT Results
Hela cell
5%
HCl + MRS pH= 4
viability
Lactic acid + MRS pH= 4
Concentration (%)
5%
viability
HNCF cell
Concentration (%)
Supernatants of: LGS: L.gasseri, LCS: L.crispatus, LGSN: LGS + NaOH, LCSN: LCS + NaOH, MRH: MRS + HCl, MRL: MRS27
+ Lactic
Acid, MRS :lactobacilli Media
Vaginal Lactobacilli & Commercial Probiotics
Supernatant MTT Result
Hela cell
viability
Commercial
Probiotics
Concentration (%)
viability
Commercial
Probiotics
pH=4
pH=5
HNCF cell
pH=6.5
Concentration (%)
Supernatants of: LGS: L.gasseri LCS: L.crispatus LRS: L.rhamnosus LPS: L.paracasei LHS: L.helveticus LNS: L.acidophilus
28NCFM
LAS: L.acidophilus candisis and MRS :lactobacilli Media as control
Homogenate, Cell Wall & Cytoplasmic Extract
MTT Results
viability
Hela cell
Concentration (%)
29
LDH & Trypan Blue Confirmed The MTT Assay Result
LCS
Live lactobacilli effect:
Inhibitory effect on Hela cells
but no effect on HNCF cells
MRS
2
5
10
Conditioned media effect:
No cytotoxic effect on both cell
lines
20
50
LDH Assay
30
Apoptotic Assay, Caspase3 Activity Assay
 LGS & LCS effect:
Caspase3 activity reduction
 MRS & MRL effect:
No change in caspase3 activity
Apoptotic inhibition of lactobacilli supernatants is independent of
pH & lactic acid
31
LDH Assay; Apoptotis & Necrosis Ratio
The ratio of LDH released from
adherent cells, floating dead
cells & the culture supernatant
were compared:
Lactobacilli supernatants
lowered the number of apoptotic
cells of Hela cells, independent of
pH & lactic acid
.
Hela cell
HNCF cell
32
Real-time PCR Results (Quality Controls)
Gel Electerophoresis; only for the first time
Bcl2: 127bp
Ladder:100bp
HPRT:131bp
Neg
Melting Point Analysis; after each Real-Time PCR run
Caspase3
MP=81°C
1.8
HPRT
MP=78°C
1.6
Bax
MP=87°C
Fas
MP=81°C
2.0
11
1.6
9
1.4
Bcl2
MP=87°C
1.8
10
1.4
8
1.2
1.2
0.8
dF/dT
dF/dT
dF/dT
7
1.0
6
5
0.8
4
0.6
1.0
0.6
3
0.4
0.4
2
0.2
0.2
1
75
33
0.0
0.0
70
6580
ºC
70
85
75
9080
ºC
85
95
90
95
0
65
65
70
75
80
deg.
70
75
85
80
90
deg.
85
95
90
95
100
Real-time PCR Results
Expression Analysis of Bax & Bcl2,
(Involved in Intrinsic Apoptotic Pathway)
Absolute Gene Regulation
Logarithmic Scale
lactobacilli Supernatant Effect on Bax Expression in Hela Cell
P= 0.35
LGS 10%
0.81
P= 0.39
LCS 10%
P= 0.66
1.02
MRS 10%
0.79
Absolute Gene Regulation
Logarithmic Scale
0.70
lactobacilli Supernatant Effect on Bcl2 Expression in Hela Cell
P= 0.39
1.12
LGS 10%
P= 0.67
1.15
P= 0.73
LCS 10%
MRS 10%
0.80
0.70
34
Real-time PCR Results
Expression Analysis of Fas,
(Involved in Extrinsic Apoptotic Pathway)
Absolute Gene Regulation
Logarithmic Scale
Lactobacilli Supernatant Effect on Fas Expression in Hela Cell
P= 0.84
P= 0.35
LGS 10%
1.02
LCS 10%
P= 0.34
MRS 10%
0.92
0.84
0.70
35
Real-time PCR Results
Expression Analysis of Caspase3,
(Activated by Both Extrinsic & Intrinsic Pathways)
Absolute Gene Regulation
Logarithmic Scale
Lactobacilli Supernatant Effect on Caspase3 Expression in Hela Cell
0.30
P= 0.31
1.36
P= 0.001
LGS 10%
P= 0.001
LCS 10%
MRS 10%
0.50
0.36
36
Biomarker Assay;
Expression Analysis of hCGb by ReaL-Time PCR
1.0
Cgb expression
placenta
0.8
+
Cervical cancer0.6
+
0.4
+
dF/dT
Tissue
Hela
0.2
Uterine myometrium
hCGb
MP= 89°C
-
0.0
Normal cervix
65
70
-
75
80
ºC
85
90
95
37
Real-time PCR Results
Expression Analysis of cgb,
(Potential Cervical Cancer Biomarker)
Absolute Gene Regulation
Logarithmic Scale
Lactobacilli Supernatant Effect on cgb Expression in Hela Cell
1.53
1.34
P= 0.001
LGS 10%
P= 0.04
LCS 10%
MRS 10%
P= 0.29
0.63
Absolut Gene Regulation
Logarithmic Scale
0.60
LGS & LCS Effect on cgb & Caspase3 Expression
P= 0.04
P= 0.29
P= 0.31
LGS
LCS
MRS
P= 0.001
caspase3
cgb
P= 0.001
P= 0.001
0.3
P= 0.001
38
Caspase3 & cgb Expression,
(Cytoplasmic Extract & Cell Wall)
No significant change was observed
39
Discussion
40
Snapshot of Results
The Prevalence of Lactobacilli in Different Grades:
(biochemical i & Multiplex PCR)
 Some lactobacilli were more prevalent in vaginal flora of healthy women
L. crispatus & L. jensenii higher in healthy women & L. iners higher in BV infected
 Bacterial Component Separation; Searching For Main Effectors
Cytotoxic Effects
Apoptotic Effects
Lactobacilli Supernatants:
 Most potent cytotoxic effects
Lactic acid production >>pH
Tumor cells >>>Normal cells
Normal 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 effect
Cell 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 independent
Intrinsic 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
41
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 antitumor activity
• Orlando et al. 2009:
Cytoplasm fraction from
Lactobacillus rhamnosus
GG induced an antiproliferative 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 Effects
Which 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
Our study:
lactobacilli supernatants have anti-apoptotic effect on Hela cells
This anti-apoptotic effect was significantly lactic acid independent.
47
(Brion et al., 2011)
Puzzle Arrangement
(Our Study & Other Studies)
Our finding:
Anti-apoptotic effect of
lactobacilli &
over-expression of hCGb
(Khailova et al.2010)
Lactobacilli
supernatant
(Iles, 2007)
(Kim et al., 2010)
(Iles, 2007)
(Tsukamoto et al., 2008)
(Del Canto et al., 2007)
Cell death
Autophagy
(Vermeulen et al.,2010)
TGF β
receptor
(Brion et al., 2011)
MKP1
48
Apoptosis Inhibits Autophagy
(Kang et al., 2011)
49
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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