Mitogen-activated Protein Kinases p38-beta Contributes to TNF-alpha Resistance in Oral Cancer Mediated BAD ser112 Phosphorylation

Figure 1. The relationships between TNF-α expressions and cell proliferation rate in normal / oral cancer cells. (A) p38 MAPK over expression induced TNF-α releasing and BAD

phosphorylation in tumor cell lines rather than normal cells lines. (B) The cell proliferation rates in tumor cell lines are higher than normal cell lines. (Cell lines: H9C2 is a rat cardiomyoblast cell line as normal. N28 is none tumor cell line from C57B mouse. T8 and T28 are 4NQO induced oral cancer cell from C57B mouse, SCC4 and CE146T are human oral cancer cell lines.)

Mitogen-activated Protein Kinases p38-beta

Contributes to TNF-alpha Resistance in Oral Cancer

Mediated BAD

ser112

Phosphorylation

Figure 2. The role of phosphorylated BAD in TNF-α resistance. (A) After 24h

5ng/ml TNF-α treatment, Caspase 3 expression increased and phosphorylated BAD

decreased in H9C2 cell. (B) The same 24 h 5ng/ml TNF-α treatment in SCC4 cell can not induce the BAD dependent apoptosis, and show the TNF-α resistance existed in this oral cancer cell line. (C) The cell survival percentage between H9C2 and SCC4 cell line in 24h TNF-α treatment. Data are expressed as mean ± SE (n=3) and * = p <0.01, ** = p <0.05, *** = p<0.001 as compared with the control group.

Figure 3. The p38α/βMAPK related BAD

phosphorylation. The p38α/β MAPK specific inhibitor CMPD1(10μM) and SB202190(10μM) were used to

block the p38α/β expressions in human oral cancer cell line SCC4 for 24h, and the down stream BAD

phosphorylation location (serine 112, 136, 155)

decreased follow the differ p38α/β inhibitions. The SB202190 treatments remarkable lowered the

p-BADser112 and p-BADser155 induced the caspase 3 cleavage in SCC4 cells.

Figure 4. The complex formed by p38α/βMAPK and different BAD phosphorylation locations. After 30 min anisomycin treatments, the

co-immunoprecipitation assay was used in complex detection between p38α/βMAPK and differ BAD phosphorylation location (serine 112, 136, 155). The result shown that,

p38α combined with p-BAD ser155 _{only and p38β combined with p-BAD}ser112 _and

p-BADser155 _{in the formed complex.}

Figure 5. The SB202190 inhibit the p-BADser112

expressions in TNF-α resistance oral cancer cells. After 24h

TNF-α (human, 5ng) or SB202190 (10μM) co-treatments, the Laser

Scanning Confocal Microscope imagination shown a partial p-BADser112

exist in cytoplasm (in degradation progress) and a partial p-BADser112

exist in nucleus. After TNF-α treatment for 24h, a large mount of

p-BADser112 _{exist in nucleus. SB202190 (10μM) and TNF-α co-treatments}

for 24h, p-BADser112 _{exist in cytoplasm only.}

Figure 6. The SB202190 treatments caused a cell cycle arrest in SCC4 cells. The cell cycle of human oral cancer cell

SCC4 arrest while A p38β specific inhibitor SB202190 (10μM) treatments from 0 to 24h. The cell cycle of SCC4 cell is major arrest at G2/M, from 9.50% (control) to 17.36% (SB202190, 10μM) within 24h.

Figure 7. The p38 MAPK dominate TNF-α release and p38β MAPK expression cause a TNF-α resistance through the downstream BAD phosphorylation. In

several published research shown that the TNF-α releasing is dominated by p38 MAPK (p38α major) activation by the outcome stress. At the same time, the stress could induce the p38α MAPK provides a BAD phosphorylation at serine 155 and p38β MAPK expression provides a BAD phosphorylation at serine 112 and 155. The serine 112 of BAD plays a gate keeper role prevent BAD dependent apoptosis caused by the protein phosphatases (such as PP2A)

dephosphorylation. Finally, the BAD phosphorylation provide a protection of human oral

cancer cell from the stress caused TNF-α induce apoptosis and promotes oral cancer a TNF-α resistance ability.

Backgrounds: When oral

cancer is diagnosed, the

three-year survival rate

prediction of this patient is

only 58% and can only be

increased to 74% after

surgery in Taiwan. Before

the squamous cell

carcinoma (SCC) formed,

hyperlosia is an initial

stage symptom induced by

EGFR over expression in a

long term inflammation.

Thus, tumor necrosis

factor-alpha (TNF-α)

releasing in inflammation

lose it original anti-tumor

function and a tumor

necrosis factor-related

apoptosis-inducing ligand

(TRAIL) resistance might

happen in mane cases. In

our pervious study

indicated p38β MAPK over

expression in oral cancer

might be associated with

TRAIL resistance through

serine 122 of BAD

phosphorylation, and

which is a gatekeeper of

BAD-mediated apoptosis.

Materials and Methods: In

this research, a cell line

T28 from

4-nitroquinoline-N-oxode (4-NQO) induced

oral cancer in C57B mouse

and human tongue

squamous cell carcinoma

cell line SCC4 were

screened in this TNF-α

resistance issue. All

proteins from cell were

analysis by immune blot

assay.

Results: TNF-α releasing is

through p38

mitogen-activated protein kinases

and TNF-α resistance exists

in both T28 and SCC4 cell

line. Further, the serine

136 of BAD

phosphorylation was

promoted by p38α MAPK

isoform and the serine 122

and 155 of BAD

phosphorylation were

promoted by p38β MAPK

and also block the

apoptosis cause by TNF-α.

A p38β MAPK inhibitor

SB202190 (10μM) was

used, the cell cycle

arrested at G2 phase from

9.5% to 17.36% within 24h

treatment in SCC4 cells.

Conclusion: Over

expression of p38β MAPK

in oral cancer indeed

caused TNF-α induced

apoptosis resistance by

BAD phosphorylation. And

serine 122 of BAD is

control by p38β MAPK.

This suggests that p38β

MAPK is a possible

anticancer target in oral

cancer therapy.

Wei-Jen Ting

, Wei-Wen Kuo

, Chih-Yang Huang

1

_{Graduate Institute of Basic Medical Science, China Medical University, Taiwan}

_{Department of Biological Science and Technology , China Medical University, Taiwan}

_{Department of Health and Nutrition Biotechnology, Asia University, Taiwan}