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

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