Cancer preventive effects of lactic acid bacteria

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1 TALAB Symposium National Taiwan Univ. Cancer preventive effects of lactic acid bacteria Hyong Joo Lee Seoul National University 1

2 Total deaths from cancer and heart disease J. Natl. Cancer Inst

3 Factor Major causes of cancer Estimated % of all cancer deaths Diet 35 Tobacco 30 Infection 10 Reproductive & sexual behavior 7 Occupation 4 Alcohol 3 Geophysical factors 3 Pollution 2 Industrial products 1 Medicines & medical procedures 1 Food additives < 1 3

4 Cancer chemoprevention with food components Lee HJ et al., Am J. Clin. Nutr.,

5 Health benefits of lactic acid bacteria (LAB) 5

6 6

7 LAB exhibited anticancer effects (1/2) 1. Colon cancer Lc. lactis ssp. Lactis Lb. plantarum Lb. acidophilus Lb. casei Lb. rhamnosus GG Bf. longum Bf. Lactis Bb12 2. Bladder cancer Lb. rhamnosus GG Lb. casei Shirota Lb. casei 7

8 LAB exhibited anticancer effects (2/2) 3. Liver cancer Lb. rhamnosus GG Lb. rhamnosus LC705 Lb. acidophilus 4. Breast cancer Lb. acidophilus Lb. paracasei Lb. helveticus Bf. Infantis, bifidum, animalis 5. Stomach cancer Lb. plantarum Lb. casei - Shirota 8

9 Mechanisms of anticancer activity of LAB Cancer 9

10 10

11 Lactic acid bacteria used for screening Lactobacillus acidophilus IAM 1084 (Laci) Lactobacillus bulgaricus KCTC 3188 (Lbul) Lactobacillus casei ssp. casei KCTC 3109 (Lcas) Lactobacillus plantarum KCTC 3099 (Lpla) Streptococcus thermophillus KCTC 2185 (Sthe) Lactoccus lactis ssp. lactis ATCC 7962 (Llac) Lactococcus lactis ssp. cremoris ML4 (Lcre) Bifidobacterium adolescentis ATCC (Bado) Bifidobacterium breve ATCC (Bbre) Bifidobacterium longum ATCC 1570 (Blon) 11

12 Fractionation into cytoplasm and cell wall Culture of lactic acid bacteria Harvest and washing Cell disruption with sonication Ultracentrifugation 90 C, 15 min Heat-killed whole cell Supernatant Precipitant Cytoplasm Cell wall Peptidoglycan 12

13 Human cancer cell lines used Cell lines SNU-1 SNU-C2A DLD1 K562 A549 A498 HT-1376 HeLa MCF7 HepG2 PC3 Origin Gastric cancer cell line Colon adenocarcinoma cell line Colon adenocarcinoma cell line Leukemia cell line Lung carcinoma cell line Kidney carcinoma cell line Bladder carcinoma cell line Cervix carcinoma cell line Breast adenocarcinoma cell line Hepatocarcinoma cell line Prostate carcinoma cell line 13

14 LAB Cell line Anticancer activity of cytoplasm ( % inhibition, 100 μg/ml ) Laci Lbul Lcas Lpla Sthe Bado Bbre Blon Lcre Llac SNU SNUC2A DLD K A A HT HeLa 16 MCF HepG PC

15 IC 50 values of cytoplasm fractions ( μg/ml ) LAB Cell line Laci Lbul Lcas Lpla Sthe Bado Bbre Blon Lcre Llac SNUC2A > >100 > SNU1 > >100 >100 >100 >

16 16

17 Cell Cycle Control CDK 2,4,6 Cyclin D, E Cyclin A Cyclin E CDK2 prb R pprb CDK2 P Cdc34 Cyclin E degradation Cyclin D CDK(4,6) G0 G1 S CDK1 Cyclin A B degradation P APC M Phosphorylated M-phase substrate G2 Cyclins A,B CDK 1,2 M-phase substrate CDK1 Cyclin B 17

18 Cell cycle analysis through flow cytometry (Llac on SNUC2A, colon cancer cells) % DNA contents G0/G1 S G2/M % DNA contents h 72 h G0/G1 S G2/M Concentration (μg/ml) Concentration (μg/ml) S phase accumulation 18

19 Immunoblotting for cyclins and CDKs (Llac on SNUC2A, colon cancer cells) - Expression of Cyclin A increased, Cyclin E decreased - No effect on CDK1 and CDK2 19

20 Immunoblotting for CDKIs and p53 (Llac on SNUC2A, colon cancer cells) β-actin β-actin -p21 CIP1 increased - No effect on p27 KIP1 and p53 20

21 Effect on CDK2 activity (Llac on SNUC2A, colon cancer cells) A; control, B; cytoplasm treated (100 μg/ml, 72 h) Inhibition of CDK2 activity, slightly 21

22 22

23 Detection of apoptotsis in SNU-1 cells Nuclear fragmentation DNA ladder A: control B: L. lac CP treated (100 μg/ml) Apoptosis induction 23

24 Effect on caspase-3 activity (Llac on SNU1, stomach cancer cells) Unit 200 caspase-3 activity (unit) hours after treatment (h) Increase of caspase-3 activity 24

25 Effect on apoptosis related proteins (Llac on SNU1, stomach cancer cells) L. Lac CP treated (100 μg/ml) Increase of p53 expression Decrease of bcl-2 expression 25

26 Cell cycle analysis (Llac on SNU1, stomach cancer cells) % of DNA contents control treated with cytoplasmic fraction of B.longum treated with cytoplasmic fration of Lc.lactis ssp. lactis 0 G0-G1 S G2-M - G0/G1 phase accumulation 26

27 Proteins related to G0/G1 Cell cycle arrest (Llac on SNU1, stomach cancer cells) Decrease of phosphorylation of prb Decrease of cyclin D1 expression Increase of p21 expression 27

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